ÑÅËÜÑÊÎÕÎÇßÉÑÒÂÅÍÍÀß ÁÈÎËÎÃÈß, 2014, ¹ 4, ñ. 42-57 Equus caballus Í.Â. ÁÀÐÄÓÊÎÂ

ÑÅËÜÑÊÎÕÎÇßÉÑÒÂÅÍÍÀß ÁÈÎËÎÃÈß, 2014, ¹ 4, ñ. 42-57
ÓÄÊ 636.1/.2:599.723: 577.2::575.174.015.3
doi: 10.15389/agrobiology.2014.4.42rus
ISSR-PCR ÌÀÐÊÅÐÛ È ÌÎÁÈËÜÍÛÅ ÃÅÍÅÒÈ×ÅÑÊÈÅ ÝËÅÌÅÍÒÛ
 ÃÅÍÎÌÅ ÄÎÌÀØÍÅÉ ËÎØÀÄÈ Equus caballus
Í.Â. ÁÀÐÄÓÊÎÂ1, À.Â. ÔÅÎÔÈËÎÂ1, Ò.Ò. ÃËÀÇÊÎ1, 2, Â.È. ÃËÀÇÊÎ1, 2
Çàêîíîìåðíîñòè è ïðè÷èíû ðàçëè÷èé â ïîëèìîðôèçìå îòäåëüíûõ ìèêðîñàòåëëèòîâ,
êàê è âèäîñïåöèôè÷íîñòü òàêîé èçìåí÷èâîñòè äî ñèõ ïîð îñòàþòñÿ íåäîñòàòî÷íî èññëåäîâàííûìè. Òåñò-ñèñòåìû, ðàçðàáîòàííûå Ìåæäóíàðîäíîé àññîöèàöèåé ïî ãåíåòèêå æèâîòíûõ
(International Society of Animal Genetics) äëÿ ãåíåòè÷åñêîé ïàñïîðòèçàöèè ñåëüñêîõîçÿéñòâåííûõ æèâîòíûõ, îöåíêè ìåæïîðîäíûõ âçàèìîñâÿçåé, êîíñîëèäèðîâàííîñòè ïîðîä, âêëþ÷àþò íåáîëüøîå ÷èñëî ìàðêåðîâ. Ïîýòîìó äëÿ æèâîòíûõ ñåëüñêîõîçÿéñòâåííûõ âèäîâ áîëåå
ïðîñòûì è ýôôåêòèâíûì ìåòîäîì ïîëèëîêóñíîãî ãåíîòèïèðîâàíèÿ ñëóæèò èñïîëüçîâàíèå
ôðàãìåíòîâ ìèêðîñàòåëëèòíûõ ëîêóñîâ â êà÷åñòâå ÏÖÐ-ïðàéìåðîâ. Íàìè ïðåäëîæåí ìåòîä
âûÿâëåíèÿ â ñåêâåíèðîâàííûõ ïîñëåäîâàòåëüíîñòÿõ ïîçèöèîíèðîâàíèÿ èíâåðòèðîâàííûõ ïîâòîðîâ ó÷àñòêîâ ìèêðîñàòåëëèòîâ. Áûëè èññëåäîâàíû àëòàéñêàÿ ïîðîäà (n = 96), ðûñèñòûå
ïîðîäû (n = 48), êàðà÷àåâñêàÿ ïîðîäà (n = 34) äîìàøíåé ëîøàäè Equus caballus. Ïðîàíàëèçèðîâàíû íóêëåîòèäíûå ïîñëåäîâàòåëüíîñòè äëÿ ôðàãìåíòîâ (îæèäàåìàÿ äëèíà/ôàêòè÷åñêàÿ äëèíà) ðàçíîé ëîêàëèçàöèè: 1250-1350/1326 ï.í. (17-ÿ õðîìîñîìà), 1250-1350/1302 ï.í.
(2-ÿ õðîìîñîìà), 1250-350/1296 ï.í. (Õ-õðîìîñîìà), 1250-1350/1287 ï.í. (20-ÿ õðîìîñîìà),
980/986 ï.í. (23-ÿ õðîìîñîìà), 800-900/900 ï.í. (24-ÿ õðîìîñîìà), 800-900/876 ï.í. (Õ-õðîìîñîìà), 800-900/871 ï.í. (18-ÿ õðîìîñîìà), 800-900/857 ï.í. (3-ÿ õðîìîñîìà), 800-900/832 ï.í.
(6-ÿ õðîìîñîìà), 800-900/823 ï.í. (19-ÿ õðîìîñîìà), 740/739 ï.í. (16-ÿ õðîìîñîìà), 580/585 ï.í.
(1-ÿ õðîìîñîìà), 550/637 ï.í. (Õ-õðîìîñîìà), 550/572 ï.í. (10-ÿ õðîìîñîìà), 500/494 ï.í. (1-ÿ
õðîìîñîìà), 490/489 ï.í. (28-ÿ õðîìîñîìà), 380/378 ï.í. (11-ÿ õðîìîñîìà), 380/377 ï.í. (21-ÿ
õðîìîñîìà), 360 (370)/364 ï.í. (5-ÿ õðîìîñîìà), 310/309 ï.í. (5-ÿ õðîìîñîìà). Ðàññ÷èòàí èíäåêñ PIC (polymorphic information content). Âûÿâëåííûå â ãåíîìå äîìàøíåé ëîøàäè ôðàãìåíòû
ÄÍÊ (99 ó÷àñòêîâ), ôëàíêèðîâàííûå èíâåðòèðîâàííûì ïîâòîðîì (GA)9C, ñîäåðæàëè ó÷àñòêè
ãîìîëîãèè ê ìîáèëüíûì ãåíåòè÷åñêèì ýëåìåíòàì. Ïðè èõ ñîïîñòàâëåíèè ñ ïðîäóêòàìè àìïëèôèêàöèè, ïîëó÷åííûìè â ïîëèìåðàçíîé öåïíîé ðåàêöèè ó ïðåäñòàâèòåëåé ðÿäà ïîðîä ëîøàäåé ñ
èñïîëüçîâàíèåì â êà÷åñòâå ïðàéìåðà ïîñëåäîâàòåëüíîñòè (GA)9C, âûäåëåíû áëèçêèå ïî ðàçìåðó
êîíñåðâàòèâíûå è ïîëèìîðôíûå ôðàãìåíòû ÄÍÊ. Îêàçàëîñü, ÷òî ôðàãìåíòû ÄÍÊ, ïîëèìîðôíûå ó ðàçíûõ æèâîòíûõ, â áîëüøèíñòâå ñëó÷àåâ íåñóò ïîñëåäîâàòåëüíîñòè, ãîìîëîãè÷íûå ôðàãìåíòàì ðåòðîòðàíñïîçîíîâ (â ÷àñòíîñòè, ERV III), òîãäà êàê êîíñåðâàòèâíûå ãîìîëîãè÷íû ó÷àñòêàì ÄÍÊ òðàíñïîçîíîâ.  èññëåäîâàííûõ ôðàãìåíòàõ ãåíîìíîé ÄÍÊ ëîøàäè, ôëàíêèðîâàííûõ èíâåðòèðîâàííûì ïîâòîðîì (GA)9C, íàáëþäàåòñÿ óâåëè÷åííàÿ ïëîòíîñòü íóêëåîòèäíûõ ïîñëåäîâàòåëüíîñòåé, ïîòåíöèàëüíî ïðåäðàñïîëîæåííûõ ê ôîðìèðîâàíèþ G4 êâàäðóïëåêñîâ, ÷òî,
ïî-âèäèìîìó, ìîæåò áûòü àññîöèèðîâàíî ñ ïîâûøåííîé àêòèâíîñòüþ ðåêîìáèíàöèé â ýòèõ ó÷àñòêàõ. Ïîëó÷åííûå äàííûå ïîçâîëÿþò ïðåäïîëàãàòü ñâÿçü ìåæäó ïîëèìîðôèçìîì ôðàãìåíòîâ
ÄÍÊ, ôëàíêèðîâàííûõ èíâåðòèðîâàííûìè ïîâòîðàìè ìèêðîñàòåëëèòîâ, è ìîáèëüíîñòüþ ðåòðîòðàíñïîçîíîâ.
Êëþ÷åâûå ñëîâà: ISSR-PCR, àíîíèìíûå ÄÍÊ-ìàðêåðû, Equus caballus, ÄÍÊ-òðàíñïîçîíû, ðåòðîòðàíñïîçîíû, íåêàíîíè÷åñêèå ñòðóêòóðû ÄÍÊ.
Ãåíîòèïèðîâàíèå ïî ìèêðîñàòåëëèòíûì ëîêóñàì ïîëó÷èëî øèðîêîå ðàñïðîñòðàíåíèå â ïîïóëÿöèîííîé ãåíåòèêå, îñîáåííî ïðè ðåøåíèè
òàêèõ àêòóàëüíûõ çàäà÷, êàê êîíòðîëü ãåíåòè÷åñêîé ñòðóêòóðû ñåëüñêîõîçÿéñòâåííûõ âèäîâ, âûÿâëåíèå ñîðòî- è ïîðîäîñïåöèôè÷íûõ îñîáåííîñòåé èõ ãåíîôîíäîâ (1). Ìåæäóíàðîäíîé àññîöèàöèåé ïî ãåíåòèêå æèâîòíûõ (International Society of Animal Genetics — ISAG) ðàçðàáîòàíû òåñòñèñòåìû äëÿ ðàçíûõ ñåëüñêîõîçÿéñòâåííûõ âèäîâ, ðåêîìåíäîâàííûå ïðè
ãåíåòè÷åñêîé ïàñïîðòèçàöèè æèâîòíûõ, îöåíêå ìåæïîðîäíûõ âçàèìîñâÿçåé è êîíñîëèäèðîâàííîñòè ïîðîä (http://www.frontiersin.org).  òî æå âðåìÿ çàêîíîìåðíîñòè è ïðè÷èíû ðàçëè÷èé â ïîëèìîðôèçìå îòäåëüíûõ ìèêðîñàòåëëèòîâ, âèäîñïåöèôè÷íîñòü òàêîé èçìåí÷èâîñòè äî ñèõ ïîð îñòàþòñÿ íåäîñòàòî÷íî èññëåäîâàííûìè. Êðîìå òîãî, ïðåäëîæåííûå òåñò-ñèñòåìû (http://www.frontiersin.org) âêëþ÷àþò íå áîëåå äâóõ äåñÿòêîâ ìàðêåðîâ.  ýòîé ñâÿçè èìåííî äëÿ æèâîòíûõ ñåëüñêîõîçÿéñòâåííûõ âèäîâ áîëåå ïðîñòûì è ýôôåêòèâíûì ìåòîäîì ïîëèëîêóñíîãî ãåíîòèïèðîâàíèÿ
42
ïðèçíàåòñÿ èñïîëüçîâàíèå ôðàãìåíòîâ ìèêðîñàòåëëèòíûõ ëîêóñîâ â êà÷åñòâå ÏÖÐ-ïðàéìåðîâ, ïîçâîëÿþùåå çà îäíó-äâå ïîñòàíîâêè ðåàêöèè îöåíèâàòü ïîëèìîðôèçì íåñêîëüêèõ äåñÿòêîâ ôðàãìåíòîâ ÄÍÊ, ôëàíêèðîâàííûõ èíâåðòèðîâàííûì ïîâòîðîì òàêîãî ïðàéìåðà (inter-simple sequence
repeat PCR — ISSR-PCR ìàðêåðû) (2).
Ñëåäóåò îòìåòèòü, ÷òî ïðèìåíåíèå â êà÷åñòâå îäíîãî ïðàéìåðà â
ÏÖÐ êîíñåíñóñíîãî ôðàãìåíòà âûñîêîïîâòîðåííûõ ïîñëåäîâàòåëüíûõ ÄÍÊ
óñïåøíî èñïîëüçóåòñÿ äëÿ ãåíîìíîãî ñêàíèðîâàíèÿ è âûÿâëåíèÿ ïîëèìîðôèçìà ðàçëè÷íûõ ãåíîìíûõ ðàéîíîâ ó ÷åëîâåêà (3). Îáíàðóæåíà äîñòàòî÷íî âûñîêàÿ ÷àñòîòà èíâåðòèðîâàííûõ ïîâòîðîâ òàêèõ ïîñëåäîâàòåëüíîñòåé íà íåáîëüøèõ ðàññòîÿíèÿõ, ÷òî ïîçâîëÿåò â îäíîé ÏÖÐ ñ îäíèì
ïðàéìåðîì âûÿâëÿòü ïîëèìîðôíûå ó÷àñòêè ÄÍÊ (4).  ãåíîìå äîìàøíåé
ëîøàäè îêîëî 36 % íóêëåîòèäîâ âõîäÿò â äèñïåðãèðîâàííûå ïîâòîðû;
ìèêðîñàòåëëèòû çàíèìàþò îêîëî 2 % ãåíîìà (5). Òåì íå ìåíåå, ñ èñïîëüçîâàíèåì ISSR-PCR ìàðêåðîâ ìîæíî ïîëó÷àòü ïîëèëîêóñíûå ñïåêòðû
ôðàãìåíòîâ ÄÍÊ, ïîëèìîðôèçì êîòîðûõ äîñòàòî÷íî íàäåæíî îòëè÷àåò îäíó ïîðîäó ëîøàäåé îò äðóãîé (6). Ðàíåå â íàøèõ èññëåäîâàíèÿõ ÷èñëî âûÿâëåííûõ â ÏÖÐ ôðàãìåíòîâ ÄÍÊ, ôëàíêèðîâàííûõ èíâåðòèðîâàííûì
ïîâòîðîì (ÔÈÏ) ìèêðîñàòåëëèòà, è èõ ïîëèìîðôèçì çàâèñåëè îò íóêëåîòèäíîé ïîñëåäîâàòåëüíîñòè ïðàéìåðà (6).  ïîëó÷åííûõ ñïåêòðàõ ïðîäóêòîâ àìïëèôèêàöèè âûäåëÿëèñü ó÷àñòêè, êîíñåðâàòèâíûå è âûñîêîïîëèìîðôíûå ó âñåõ èññëåäîâàííûõ ëîøàäåé.
Äëÿ òîãî ÷òîáû îöåíèòü âîçìîæíûå ïðè÷èíû îïèñàííûõ ðàçëè÷èé ïî êîíñåðâàòèâíîñòè è ïîëèìîðôèçìó, ìû ñîïîñòàâèëè äàííûå ïî
ýêñïåðèìåíòàëüíî íàáëþäàåìîìó ïîëèìîðôèçìó ëîêóñîâ è ïåðâè÷íîé
ñòðóêòóðå ó÷àñòêîâ áëèçêîé äëèíû, ôëàíêèðîâàííûõ èíâåðòèðîâàííûì
ïîâòîðîì ìèêðîñàòåëëèòà, êîòîðûé èñïîëüçîâàëñÿ â êà÷åñòâå ÏÖÐ-ïðàéìåðà, â ñåêâåíèðîâàííûõ ïîñëåäîâàòåëüíîñòÿõ ãåíîìà ëîøàäè èç GenBank (http://www.ncbi.nlm.nih.gov/).
Ìåòîäèêà. Èññëåäîâàíèÿ âûïîëíÿëèñü íà ãåíîìíîé ÄÍÊ ñëåäóþùèõ ïîðîä äîìàøíåé ëîøàäè Equus caballus: àëòàéñêàÿ (48 îáðàçöîâ èç õîçÿéñòâà «Ýí÷è» è 48 îáðàçöîâ èç ÑÏÊ «×èíãèç», Àëòàéñêèé êðàé); ãðóïïà
ëîøàäåé ðûñèñòûõ ïîðîä, ñîäåðæàùèõñÿ â êîíåçàâîäå «Ïîâîðîò Â.Ï.»
(Ìîñêîâñêàÿ îáë.) (7 îáðàçöîâ îò àìåðèêàíñêèõ ðûñàêîâ, 6 — îò ðóññêèõ
ðûñàêîâ, 4 — îò îðëîâñêèõ ðûñàêîâ, 31 — îò ïîìåñíûõ ðóññêèõ ðûñàêîâ,
óëó÷øåííûõ àìåðèêàíñêèìè ðûñàêàìè); êàðà÷àåâñêàÿ ïîðîäà (34 îáðàçöà).
ÄÍÊ âûäåëÿëè èç îáðàçöîâ ïåðèôåðè÷åñêîé êðîâè ñ ïîìîùüþ íàáîðà ÄÍÊ-Ýêñòðàí 1 («Ñèíòîë», Ðîññèÿ). Äëÿ èññëåäîâàíèÿ ãåíåòè÷åñêîé
ñòðóêòóðû è ïîëèìîðôèçìà âíóòðè âûøåóêàçàííûõ ïîðîä ëîøàäåé ïðèìåíèëè ïîëèìåðàçíóþ öåïíóþ ðåàêöèþ (ÏÖÐ) ISSR-PCR (inter-simple
sequence repeat PCR), â êîòîðîé â êà÷åñòâå ïðàéìåðà èñïîëüçîâàëè íóêëåîòèäíóþ ïîñëåäîâàòåëüíîñòü (GA)9C. ÏÖÐ âûïîëíÿëè â àìïëèôèêàòîðå
Òåðöèê («ÄÍÊ-òåõíîëîãèÿ», Ðîññèÿ) â ñëåäóþùåì ðåæèìå: ïåðâè÷íàÿ äåíàòóðàöèÿ (94 °Ñ, 2 ìèí); äåíàòóðàöèÿ (94 °Ñ, 30 ñ), îòæèã (55 °Ñ, 30 ñ), ýëîíãàöèÿ (72 °Ñ, 2 ìèí) — 35 öèêëîâ; ôèíàëüíàÿ ýëîíãàöèÿ (72 °Ñ, 10 ìèí).
Äëÿ ïðîâåäåíèÿ ÏÖÐ èñïîëüçîâàëè íàáîðû ðåàãåíòîâ PCR Core («Áèîêîì», Ðîññèÿ) è ÏÖÐ-ÐÂ (ÏÖÐ â ðåàëüíîì âðåìåíè) («Ñèíòîë», Ðîññèÿ).
Ïðîäóêòû àìïëèôèêàöèè, îêðàøåííûå â ðàñòâîðå áðîìèñòîãî ýòèäèÿ, ðàçäåëÿëè â 1,5 % àãàðîçíîì ãåëå ñ ïîñëåäóþùåé âèçóàëèçàöèåé íà òðàíñèëëþìèíàòîðå ÓÂÒ-1 («Áèîêîì», Ðîññèÿ). Äëèíû ïîëó÷åííûõ ôðàãìåíòîâ îöåíèâàëè ñ ïîìîùüþ ìàðêåðà ìîëåêóëÿðíûõ ìàññ M 27 (100 bp + 1,5 Kb + 3 Kb,
12 ôðàãìåíòîâ îò 100 äî 3000 ï.í., «ÑèáÝíçèì», Ðîññèÿ).
Èíäåêñ PIC (polymorphic information content) âû÷èñëÿëè ïî ôîðìó43
ëå: PIC = 2f (1 − f), ãäå f — ÷àñòîòà îäíîãî èç äâóõ àëëåëåé, ðàññ÷èòàííàÿ
êàê f = √R, ãäå R — ÷àñòîòà âàðèàíòîâ, ó êîòîðûõ îòñóòñòâîâàë ôðàãìåíò
ÄÍÊ ñîîòâåòñòâóþùåé äëèíû (ãîìîçèãîòû ïî «ðåöåññèâíîìó» àëëåëþ).
Ðåçóëüòàòû. Ý ê ñïå ðè ìå íò àëü íûå ñ ï å êò ðû Ä ÍÊ-ôð à ãì å íòî â
Ô ÈÏ (GA)9C ó è ñ ñ ë å ä î â à í í û õ ï î ð î ä ë î ø à ä å é. Íàïîìíèì, ÷òî
ISSR-ìàðêåðû õàðàêòåðèçóþòñÿ äîìèíàíòíûì òèïîì ïðîÿâëåíèÿ. Â
ñïåêòðàõ àìïëèêîíîâ, êîòîðûå ìû ïîëó÷èëè ñ ïðàéìåðîì (GA)9C â
ISSR-PCR (òàáë. 1) íà ãåíîìíîé ÄÍÊ ëîøàäåé ðàçíûõ ïîðîä, ñóììàðíî
ïðèñóòñòâîâàëè 20 ôðàãìåíòîâ (äî 20 000 ï.í.), ïðè ýòîì 11 èç íèõ îêà1. Çíà÷åíèÿ èíäåêñà ïîëèìîðôíîãî èíôîðìàöè- çàëèñü ïîëèìîðôíûìè õîòÿ
îííîãî ñîäåðæàíèÿ ëîêóñà (PIC) ó èññëåäî- áû ó îäíîé èç èññëåäîâàíâàííûõ ïîðîä äîìàøíåé ëîøàäè Equus cabal- íûõ ïîðîä. Ïðè ðàñ÷åòå èílus ïî ðåçóëüòàòàì ISSR-PCR
äåêñà ïîëèìîðôíîãî èíôîðìàöèîííîãî ñîäåðæàíèÿ ëîÄëèíà
PIC ïî ïîðîäàì
ëîêóñà, ï.í. ðûñèñòûå àëòàéñêàÿ êàðà÷àåâñêàÿ ïî âñåì êóñà (PIC) êàæäûé ôðàãìåíò
1880
0
0
0
0
ÄÍÊ ðàññìàòðèâàëè êàê îò1700
0
0
0
0,43
äåëüíûé ëîêóñ. Íàèáîëüøèé
1600
0,49
0
0
0,38
1580
0,27
0,19
0
0,11
ïîëèìîðôèçì îòìå÷àëè ó ëî1400
0
0
0
0
øàäåé àëòàéñêîé ïîðîäû,
1300
0
0
0
0
1200
0
0
0
0
íàèìåíüøèé — ó êàðà÷àåâ1100
0,43
0,50
0
0,42
ñêîé. Ïîëèìîðôíûìè îêà980
0,41
0,48
0
0,49
950
0
0
0
0
çàëèñü ôðàãìåíòû ÄÍÊ 1700,
900
0
0
0
0
1600, 1580, 1100, 980, 880,
880
0
0,43
0
0,29
840
0,43
0,35
0
0,29
840, 740, 550, 500 è 360 ï.í.;
7401
–
0,44
–
–
êîíñåðâàòèâíûìè — 1880,
580
0
0
0
0
550
0
0,35
0,28
0,29
1400, 1300, 1200, 950, 900,
500
0,43
0,35
0,47
0,44
580, 490, 390 ï.í. Ïðè ýòîì
490
0
0
0
0
390
0
0
0
0
ïîëèìîðôèçì êàæäîãî ëîêó3601 (370)
0,47
0,40
–
–
ñà â çíà÷èòåëüíîé ñòåïåíè
PICñð.
0,14
0,15
0,04
0,17
Ï ð è ì å ÷ à í è å. Ïðî÷åðêè îçíà÷àþò, ÷òî ó êàðà÷àåâñêîé ëî- âàðüèðîâàë â çàâèñèìîñòè
øàäè íå ó÷èòûâàëè ôðàãìåíòû ìåíüøåé äëèíû, ÷åì 390 ï.í.; 1 îò ïîðîäíîé ïðèíàäëåæíî(â âåðõíåì èíäåêñå) — îïóáëèêîâàííûå äàííûå (6).  êà÷åñòâå
ñòè æèâîòíûõ.
ïðàéìåðà èñïîëüçîâàíà ïîñëåäîâàòåëüíîñòü (GA)9C.
Ðàñ÷åòíûå ñïåêò ð û Ä Í Ê - ô ð à ã ì å í ò î â Ô È Ï (GA)9C â ñ å ê â å í è ð î â à í í û õ ï î ñ ë å ä î â à ò å ë ü í î ñ ò ÿ õ ã å í î ì à ä î ì à ø í å é ë î ø à ä è (Equus caballus, GenBank). Äëÿ âûÿâëåíèÿ âíóòðåííåé ñòðóêòóðû ïîëó÷åííûõ â ðåçóëüòàòå
ISSR-PCR ïîëèìîðôíûõ è ìîíîìîðôíûõ ôðàãìåíòîâ ÄÍÊ, ôëàíêèðîâàííûõ èíâåðòèðîâàííûì ïîâòîðîì (GA)9C, áûëè ðàçðàáîòàíû àëãîðèòìû
íà ÿçûêå Python 2.7.3 (http://www.python.org/):
import re
f=open("chromosome1.txt", "r")
for line in f.readlines():
print
re.findall(r"......AGAGA[^...]{0,4}GAGAGAC[^...]{50,2000}GTCTCTC[^...]{0,4}TCTCT......", line)
f.close(),
ãäå chromosome1.txt — ôàéë èç GenBank (http://www.ncbi.nlm.nih.gov) äëÿ õðîìîñîìû ñ óäàëåííûìè â òåêñòîâîì ðåäàêòîðå ïðîáåëàìè è ïåðåíîñàìè
ñòðîê (â àíàëèç âêëþ÷àëè ïîñëåäîâàòåëüíî âñå àóòîñîìû è Õ-õðîìîñîìó
ëîøàäè); ......AGAGA[^...]{0,4}GAGAGAC — ïðÿìàÿ ïîñëåäîâàòåëüíîñòü
äëÿ ïðàéìåðà (GA)9C, ãäå òî÷êàìè óêàçûâàëèñü íóêëåîòèäû, êîòîðûå ìîãëè áûòü íåñïàðåííûìè; [^...]{0,4} — ñèìâîë, êîòîðûì çàäàåòñÿ øàã äëèíîé
íå áîëåå 4 íóêëåîòèäîâ ïîñëå æåñòêî äåòåðìèíèðîâàííûõ 7 íóêëåîòèäîâ ñ
3´-êîíöà ïðàéìåðà; GTCTCTC[^...]{0,4}TCTCT...... — èíâåðòèðîâàííàÿ ïîñëåäîâàòåëüíîñòü ïðàéìåðà â òîé æå öåïè ÄÍÊ; {50,2000} — óêàçàíèå íóê44
ëåîòèäíîãî ïðîìåæóòêà ìåæäó ñàéòàìè îòæèãà ïðàéìåðà. Àëãîðèòì ïîèñêà
âîçìîæíûõ àìïëèôèöèðóåìûõ ïîñëåäîâàòåëüíîñòåé ÄÍÊ ïðåäïîëàãàë, ÷òî
íå ìåíåå 7 íóêëåîòèäîâ ñ 3´-êîíöà ïðàéìåðà äîëæíû îòæèãàòüñÿ òî÷íî è
íå ìåíåå 12 íóêëåîòèäîâ — èìåòü êîìïëåìåíòàðíîñòü ê ïðàéìåðó (GA)9C.
Òàêîå óñëîâèå îñíîâàíî íà ýêñïåðèìåíòàëüíûõ èññëåäîâàíèÿõ (7, 8) îá
èñòî÷íèêàõ îøèáîê ïðè îòæèãå ïðàéìåðîâ â ÏÖÐ.
Äëèíà ïîñëåäîâàòåëüíîñòè ìåæäó ïðåäïîëàãàåìûìè ñàéòàìè îòæèãà ïðàéìåðîâ ïî óñëîâèÿì ïîèñêà ñîñòàâëÿëà íå ìåíåå 50 è íå áîëåå 2000
íóêëåîòèäîâ. Äî íà÷àëà ðàáîòû ñ ñèêâåíñàìè õðîìîñîì âñå ñòðîêè íåîáõîäèìî áûëî îáúåäèíèòü â îäíó, ÷òî âûïîëíÿëîñü ïðè ïîìîùè òåêñòîâîãî
ðåäàêòîðà èëè ïðîãðàììû BioEdit, ïðåäñòàâëåííîé â ñâîáîäíîì äîñòóïå
(http://www.mbio.ncsu.edu/BioEdit/bioedit).
 êà÷åñòâå âíóòðåííåãî êîíòðîëÿ ñ èñïîëüçîâàíèåì òåõ æå ïîäõîäîâ âûäåëÿëè è ðàññìàòðèâàëè ôðàãìåíòû ñåêâåíèðîâàííûõ ïîñëåäîâàòåëüíîñòåé ÄÍÊ, íà ôëàíãàõ êîòîðûõ ëîêàëèçîâàíû ïðÿìûå ïîâòîðû ïîñëåäîâàòåëüíîñòè (GA)9C.
Ñðàâíåíèå ñïåêòðîâ ýêñïåðèìåíòàëüíûõ è ðàñ÷åòíûõ
ô ð à ã ì å í ò î â ÄÍÊ Ô È Ï (GA)9C â ã å í î ì å ä î ì à ø í å é ë î ø à ä è.
Ýëåêòðîôîðåòè÷åñêèå ñïåêòðû ïðîäóêòîâ àìïëèôèêàöèè ó÷àñòêîâ ÄÍÊ
(àìïëèêîíîâ) áûëè ïðåäñòàâëåíû çîíàìè ñ ðàçëè÷íîé èíòåíñèâíîñòüþ
îêðàøèâàíèÿ, ÷òî äîïóñêàåò íàëè÷èå â íèõ ôðàãìåíòîâ ÄÍÊ ñ áëèçêèìè
äëèíàìè. Óñëîâíî ìû îáîçíà÷èëè çîíû êàê ìàæîðíûå (íàèáîëåå êðóïíûå
è èíòåíñèâíî îêðàøåííûå) è ìèíîðíûå. Äëÿ òîãî ÷òîáû óòî÷íèòü âîçìîæíîñòü ïðèñóòñòâèÿ â ìàæîðíûõ çîíàõ íåñêîëüêèõ ôðàãìåíòîâ ÄÍÊ,
áûëè âûïîëíåíû èññëåäîâàíèÿ, â êîòîðûõ íà îäíîé è òîé æå ãåíîìíîé
ÄÍÊ ïðîâîäèëè ÏÖÐ ïðè ðàçíûõ òåìïåðàòóðàõ îòæèãà.  ñïåêòðàõ àìïëèêîíîâ, ïîëó÷åííûõ ñ ïðàéìåðîì (GA)9C (ðèñ., À), âûäåëèëèñü äâå
êðóïíûõ çîíû â ðàçìåðíîì äèàïàçîíå ïðèìåðíî 800-900 ï.í. è 12501350 ï.í., êîòîðûå ïðè ïîâûøåíèè ñïåöèôè÷íîñòè ÏÖÐ çà ñ÷åò óâåëè÷åíèÿ òåìïåðàòóðû îòæèãà äî 62 °Ñ ïðåâðàùàëèñü â êëàñòåðû, ñîñòîÿùèå èç
ãðóïï àìïëèêîíîâ, è ñìåùàëèñü ââåðõ (ñì. ðèñ., À).
Ïðèìåð ñïåêòðà àìïëèêîíîâ ISSR-PCR ñ ïðàéìåðîì (GA)9C äëÿ ãåíîìà äîìàøíåé ëîøàäè Equus
caballus ïðè ðàçíûõ òåìïåðàòóðàõ îòæèãà (À) è èñïîëüçîâàíèè ëèîôèëèçèðîâàííîé ïîëèìåðàçû
(Á, òåìïåðàòóðà îòæèãà 55 °Ñ): 1, 2 — 55 °Ñ; 3, 4 — 62 °Ñ; Ì — ìàðêåð ìîëåêóëÿðíûõ ìàññ
M 27 («ÑèáÝíçèì», Ðîññèÿ); à, á — ñîîòâåòñòâåííî êëàñòåðû 1250-1350 ï.í. è 800-900 ï.í.,
â — áýíä ~ 590 ï.í., ã — âåðîÿòíûé ãåòåðîãåííûé áýíä ~ 559 ï.í., ä, å — ìàæîðíûå áýíäû
ñîîòâåòñòâåííî ~ 490 ï.í. è ~ 380 ï.í. 45
Ñîïîñòàâèâ äàííûå î ïîñëåäîâàòåëüíîñòÿõ ÄÍÊ, íàéäåííûõ â ñåêâåíèðîâàííûõ õðîìîñîìàõ ëîøàäè ñ èñïîëüçîâàíèåì îïèñàííûõ âûøå àëãîðèòìîâ ïîèñêà, ñ ðåçóëüòàòàìè, ïîëó÷åííûìè ïðè ýëåêòðîôîðåòè÷åñêîì
ðàçäåëåíèè ïðîäóêòîâ àìïëèôèêàöèè, ìû âûÿâèëè ñîîòâåòñòâèå ýêñïåðèìåíòàëüíûõ è ðàñ÷åòíûõ äëèí äëÿ ìíîãèõ ó÷àñòêîâ.
Òàê, äëÿ êëàñòåðà 1250-1350 ï.í. ôðàãìåíòàìè ÄÍÊ ñ âûñîêîêîìïëåìåíòàðíûìè ïðåäïîëàãàåìûìè ñàéòàìè îòæèãà ïðàéìåðîâ áûëè ïîñëåäîâàòåëüíîñòè 1326 ï.í. (ëîêàëèçîâàíà â 17-é õðîìîñîìå, èìååò êîìïëåìåíòàðíûå ñàéòû îòæèãà ïðàéìåðîâ ïî âñåé äëèíå ïîñëåäíèõ), 1302 ï.í.
(2-ÿ õðîìîñîìà), 1296 ï.í. (Õ-õðîìîñîìà), 1287 ï.í. (20-ÿ õðîìîñîìà).
Ðàçäåëåíèå çîí íà êëàñòåðû ïðè óâåëè÷åíèè òåìïåðàòóðû îòæèãà ïðàéìåðà ñîîòâåòñòâîâàëî ïîâûøåíèþ òî÷íîñòè îòæèãà â íàéäåííûõ ôðàãìåíòàõ
ÄÍÊ (ñì. ðèñ., òàáë. 2).
2. Ôëàíêèðîâàííûå èíâåðòèðîâàííûì ïîâòîðîì (GA)9C íóêëåîòèäíûå ïîñëåäîâàòåëüíîñòè â ñåêâåíèðîâàííîì ãåíîìå äîìàøíåé ëîøàäè Equus caballus, ñîîòâåòñòâóþùèå ðàçìåðó àìïëèêîíîâ â ISSR-PCR, è èõ ïðåäðàñïîëîæåííîñòü
ê ôîðìèðîâàíèþ G4 êâàäðóïëåêñîâ
Ä ë è í à ô ð à ã ì å í ò à Ä Í Ê 1250-1350/1326 ï.í. (17-ÿ õðîìîñîìà, PIC «−»)
GGAAGAGAGAGAGAGAGACAGACCGTGGTCATTTGATCCCTGAATGTAGGTTTGCTGAG
ACCAGCACTCAGTCTGACTCCTCAGTTATGTGAGCCAGCATATTACTTTTCTTCATAAA
GAGGTTTGAGTGGGTTTCTGACACTGGCACTGAGAGAGTCCTGAGTAACTCAAATATGG
AAATAAATGGAAATTATACCAAATGAAGACATATGTAAAAAGGAGAACTTCCTCTGATG
TGTGCAACAGTGAAAAAGCTCATCAAATCTGAAGCAGCTCCGAGACAGCTGAAGTCGAC
AGATCCTAGGGTCTTTACCATAATCATACTAATTTTGTATATGTGATGCTTTAAAAATA
TCACACATATTCTTTACAAGTATAATTTTTTAATTTATTTTTTCTTCTCCCTTTTAAAG
AAGGAGATTCTAGAATACAAATGGAGCTCAAGGAATATATCATCCTTTCAGCATGCCTT
AAAGTTCTGTATCAAACAATGAATTATTGTATGTGAAAGGGGTTTATGTTTCTGAAAAT
TATTATAATAGGGATTATTTAAAATATGATTTTGAAGCATTAACATATTTACTTATTTG
CACCCTTTTATATATTAGTTTTTATATTTAAAAGAAATGTAGCCACATTTCTTCAGTAA
TATTGAACAGTTGAAAAGTTTACCATCATCCTGGACTTTTCCTCATTATTGGGAAGTAG
AGGAATTTTACTGTAATATATTTCAATAATGTTTATCTAGTGTTAGCTTTTGAGGTTCA
AAGTTTTAACCCCAGAGAAGATCTTTTTTATGTACATGGTACTTGAAAATGCAATCATG
ACTGAAATGAGTTAATCTAAAACTGTGAAACAAGAATTAATCACTTATTTGTCAAGATC
AAGACATGATGTGTCAATTTATTTAGCACACAGGAGGGCCATCCCTATACATACTAAAA
CATATTTGGAGGATTGTTAGAAATCAACTATATTTGATGGAAACATTTCTGCAAAATTA
GCTAAATATAATACTGAATAGTTTGAGCTTCTATTTGCTTTTAAAATAGTAAATCTCTT
TTGGTTGACCACAGATATCAATTATAATTATCAGTTACTGTGTAATAGGTTGATGCTCT
TTATTGCTTTTTATGCATGTAAGTTCACTTGTCTCAATTTTAAATGATTTCAGACTGGC
GTGTTTTTGAAGTGTTTACTTTCATAGTTTCTATCTCCTTCTTTACTGTTGAAATCCCA
GCTGTCACTTCTATTTCTGTCAATATGCTAGCCTTCAGCAATCTCTCAATTCCTTCTTG
TTCTTCTTGGTCTCTCTCTCTCTTTCTC
Ä ë è í à ô ð à ã ì å í ò à Ä Í Ê 1250-1350/1302 ï.í. (2-ÿ õðîìîñîìà, PIC «−»)
GAAAGAGAGAGAGAGAGACAGGTTCTTTGTGCATTTCGCATCCTGTTAGTGGGTGTGGG
GGTGCATGGTTTATGTTAGTTACATGGTTATGAGGGTGACAACCTGTGTCCCTGTCTCA
TACCGTCTGGGGATATCATGTGCCCGTCTCACGATCTGGGCGAAGTGACAGGTTATATG
CTTAGCTCATCCTGTTAGGAGGGTGGTCACTGACTTGTGTGCTTGTCTCTGGCTGTTGG
GTATGTGTGAGGTGTCACCATGTGCCCGTCTGATGTTGTGAGGAGTGACATGTGTGATT
ATCTCGTGCTGTTGGGAGGTTGTCAGTAACTTGGGGGTTGTCTCTCACCCTAAAGAGAC
AACATGTTGCCCAGTGGTGTGGGGCTGCGGTTGGCAGGGCCTTCCCACGCAGACCACAG
GGCCCCAAAGGGCTGGCCAGAGGAAGCTCTGGGGGCCCTGAGTACAGGGACAGATGTGG
GCTGGAGCGAGGGTCTCAGTCCCTCATGACCTACCTTCTGGCCCAGGTGCCAACCCTTG
AGACTGTTCCCCAGGAGGAAGGACCCCGTGTATCTTCACACAGTGTGGAAAGGGGCCCC
TGGCTCTGGGGCAGGTGTGACTGGGGAGGTAGAGCACCAGGCGGGCTTCACATGGGGGG
CTGTGTCCCCGCAGGCCTGGGGGGCCCTATTCCCTGCCTCTTCTGCCCCTGAAACTGTT
AAACTAATTAAGGCTGTGGAGCCGCAGGATTAATCGGGGCAGCTCGGCGCGGGCCGGGA
GCTCAGGTAGTCGATTAGTGTGCCGCGGAGGATATTGGATTTCTGAACCGCAAGTCAGC
ACTATTCGGGCCTGTTATCTCTCCGAGGCTTGAGGGTCAGGAGGCGGGAGCCTCCGGGC
GCACTGGCCGCCCCCGCCCGGCCAGCTCTGGAGCGTCCACTCCCCCTGCCCACACACCG
CCAGCCTGTCTGCCTGGCATCCCTACCCACTCCCCCCTGCCCTCCTGGCCCCACCGCTG
CCCACACGGCACCCTCACCCAGCCCTTCTCCCTGGCTCACCCTGGCCCTACAGTCATGT
TCTCTCTCCTTCTTGTTCCCTCCTGGCATGACTCCCACTCTGTGCCTCCCTGACACCTG
CTCCTCAGCTTCTCACCAGCCTCTCTCCACTTTGGTCTCCATCTCCCTGTCACTGTCTC
TGTCCCCCAGTCTCTGTTAGCCTCTCCTATCACCACTCTCTGTTTTTGTGTTTGTGTTT
GTGAGTGCCTGTCTCTTACAGTTTCTGTCTGTCTCCATCTCTCTGTCTCTCTCCCTCTG
TCTG
Ä ë è í à ô ð à ã ì å í ò à Ä Í Ê 1250-1350/1296 ï.í. (Õ-õðîìîñîìà, PIC «−»)
GGATGAGAGAGAGAGAGACATGAATGACAGAGTAAGAGAGACAGATGATGAATAAGTAG
ATGACAGAGAGAGATAGATAATAGCTTATTTACTAATTTATTTAGTCATTTACTGTGGC
AGACTCCATACTAGATGCTGAGGGCGCAGAGGTGACTAAGATAAGGGTCCCTGTTTTCA
ACAAGCTTGTTACATAGTAGGATAAATCAGCTGTCTACCTCCTAGGAGCAGTAGTGGAC
AGACTCAGGCTGGATTCCATCCCAGCTAGGAATGCCCCTCACCCCAGCGGAAACACAGA
AAATCCCAGCAGTGCAAGGTTCTAGAGGGCTAAGGCCTAAAAAGGTAGTCCTGAAGAGA
ACGGCGTTGCTGGGAAAAACCTGGGGTAGAATGGTAACAGGCCAGGCACAGACAAGGAG
46
Ïðîäîëæåíèå òàáëèöû 2
GTGTAAGAGGAAGAACAGGGGAAGCTGCTTGTCAAAGGTCAGGAACCAGAGCCACAGGT
CAGCAGCAGTGGCTAAGATTTATGGGAAGGAAAGCCAGGTGAGGCCCCCACTTCTAACC
ACATTATTCTGTTATGGATTTCAGAGGGCCCAGCAGGGACGCTGAAGGCATGAGAAAGC
CTTGGGGGTGTGTAACTGCTCCAGAGTCTCACACACAAAGCCATGATGTTAGAAATATC
TCCAGCTGCTGTTACCCGGTAGTCTTCAACAACTATTTATAAAACCTCTCGCAGAAGTA
CTATGTACTACTACGCACTGTGCCATGTGTATCTACTATATACTATGCTTCTAGGCACT
GAAGATACAGTACAGAACAGGGTATGTCTCTTATTTTGGGCCCTTCTTCTACATCCAGG
ATTCCTGAAGTTCTCCTGAGCCCAGGGTTTAAAATGTGAGCAGTCAGTGTTGATACCAC
CTTCTCCCCAACTGTCCAATCCTTCACTGAGTTCTGCTGAGTTTTCTTTTATGATATGT
CTCTGGCTTTGCCTCTCTCTCTCCATTCTCAGTGAAACTATTTCACATAATTGCAGTTC
TCCTCCACAGAATCCATCACCAACACTTCCTATTCTAGTCCAAGCCCTTAACATTCTAT
GTCCAAAATACTGCATCAGTATGCCAGCCAGTGTCCCCTTGTTCCACCTCCAGTCACTG
CTTTTAGACCATATCATGTCAAACACCAATTGAATTGTGGCACTCTCCTATTCAAGAAT
ATTCACTGGCTCCCTATTTCTTACCCTGTCCTGCATCAAATGTAAGCTGGATTAGACCT
TCAAAGCCCTCTATAAGCTATCTTTATCCGCCAGCCCAGTCTCTCACATCTCTGCTC
Ä ë è í à ô ð à ã ì å í ò à Ä Í Ê 1250-1350/1287 ï.í. (20-ÿ õðîìîñîìà, PIC «−»»)
GGAGAGAGGAGAGAGAGACACAGAGAGAGAAAGACTGAGAATATTGCAGGTATTCCTAG
CTGCCTAAATTTTCTGCTCAAAAATATAGGAGAACCCGCAGCCCGGTGGAAGGAGCCTA
TTTAAGTCCGGAGGAAGGTTGAACTTTAACAGAAATGGCAGTCAGGCCAGTCGAACGGG
TTCCTGAAACATCCAAAAAGGTTTTCTGTTGCCCCTGGCTTTGCCTGGAAAGGATAAGT
TTAGTGTACTTGAAGATTGATTGCATGTATTGTAAATTGAGGTAAAAATCAGACTGTAT
GAGCACTAAAGAGGGAAAGGCGGCACACGGATGAAAAACCCCGGCTCGCCTCTGTCCTT
TAATAAATAAGCATAATCTCCGATATTCAGTGAAAGTCCGCAGACGTCACTGTAAACAA
CCGGGCCCACCGGGTTACTTTTCTCTCTCCTCCAAATGATGAGGGAACGCCTTTTTATT
TTGTTCTTAATCTGCACACAAAGCACCTAGGGCGATCGACTCTTCCCATTAGGAAAGTC
CCTCTCCAGGAACAAGGACACCTACGTTCTCTACCAGCCCCCAGCGATGCTCTCCCTCC
CTGCCTTGTCCCGGGGAGCAGGGGCTGCTTTACGCGTGTCCCCGGCTCAGTTAATTGAA
GTTCTGCCTCTTTCCTGCTAAACTTTTTCCCCAGTCTGCCTTTGCCAGCCCCCTGGTCT
GATTACATCTAGTAATTCTCCACTGAATGAACGTCATTTACAATAGTAGGGGAGCTCTG
CGCTCGCTGCTGCTTCACGCTCCATTTGTCCTCCATTCTCCCTTTAAAGTACTCGTGTA
ATATTAATAGTCATGAATATCAATTATTATGAGGCGGTGTAGGCAAGCGGAGGGAGGAA
GGGGCGCCTGAGCAGTCTGAGCCCAGCTTCGGGTGGAAGAGGACTGCGTCTGGAGCCCC
GAAGAACGGCAGAAGAAAAAAGGCCAAAAAGACAGCAAGAGACGGAGAAACAGCTCTGT
CTGTGTGTGACAGGCATCGCCAATCTTGTTTGGGAGGGGGGGGGGGGTAAATTTTTTTT
TTTGATGGCTTGTCCTGGAAACACCTCAAGCTCGGCTCCTGTGTCCAGCTCGTTGCTCA
GCTGGACTCCTTGATTGTTTTTTTTTAATCATTGTTTGATTTTGAGCAGTAACCAGGCT
TTTTTTTCCAGATGTTAGTCCACACCTATTCATCCATGGTGAGTTTGGATTCACGTTGT
ACCAGAATTGCATGCTCCCTCCTCTCACTGTCTCTCTGTCTCTCTGTC
Ä ë è í à ô ð à ã ì å í ò à Ä Í Ê 980/986 ï.í. (23-ÿ õðîìîñîìà, PIC = 0,49)
TGGGGAGAGAGAGAGAGACAGTGGCTTCTTTCATCTGATGGGTATCTGCTCTAATAGCA
GTATCCAGTGAATACCTAGCTTCGATGGTTCGTTTCACCTGCCTCTCTCTGAGTGGGTA
AAAGGGTATCACAGAAAAGAAGTATACATACCTTATATATCTCATAAGTTTGGTTATAG
CACAGAATGTAAATGCTATCATGAAACTCACCCAGCTTGTATTTGGTGTATTAGAAAAC
TATCTCTCCAGAGGAGAAAAACAAACAAAAACATTCTGCGCTGAAATATGGTAGAAGAC
ACTTGGATAAGCAAAGGTAAAGGCTTTCTTTACCCTGGGACTTCTCAGAGTGGACTTGG
GGCTAAAACCCAGGTCTCTTGCTTCAGAATAATTGCCTTTCCCCCCCTCACAATGCTGC
TCTGTTTAGTTCATAGGGCCCAAGGCCCTCAGTTTGAGGCCCAAACCAGCGGCTTTGAG
AAATTATGCCCCCTCATCCTGATCATCACAAATTCAAGGTTGCTGACGAGGGAAGAAGG
AGAAGCCAGGGGAACAAGAATAGAGTGGAAATACAGGGGTTAAGGGTATTTCCTCTGGA
GTCAGAAGGTACTGAATGTTTATCCCCATCTGACACTAGCTTTGTGAACTTGGATCAAG
AAATTTGTTAAGGTTCAGCTTCCTCATCTTTAATACAGAGTTGTTTAAAGAACAAAATA
TGAGAGTTCTTTTTCAATGTTACTGTTACTATTTTTTATTAATTTTTGTCGAGTCTAAC
AACCCATCCCTTTTTCAATGGGGTTATCAGTTTCATCTCCAATTATGAGTAGCAGCTCT
TTCATTGGTTTCCTTAAAATGGTATCAAGGAAAAAGAAATATGCCTTAGAAGAAATTGA
GAGGCTTTATATCTAAATGCACTGTATTAAAACAGTCTCAAAGTAATTAACGTTATTTT
AATTAAAGTATGCAAATCTCTCTGTCTCTCTGTCTCTAACAC
Ä ë è í à ô ð à ã ì å í ò à Ä Í Ê 800-900/900 ï.í. (24-ÿ õðîìîñîìà, PIC = 0)
TAGAAAGAGAGAGAGAGACCACCAGGGAATTGTGAGGAAAAGAAAAATGAGGGAAATGT
CTCGATTTAATAGGAATTTGGGAGTGTTGAGAGCATTTTAAGAAAAATGCTTGCTTTTT
CACTAGATGAGTCATTTTGTTCAAAGGGAGAAGAAACTTACTACTTTCTCTGAAGTGTA
ATAGAACTGCAGACCTAGCTCCACAACTTTTTTCTCTTTTACATAATCTTGTCCTTATT
CAAGAGCTAGTGAGTGAGGGTTTTCCCATGAGAACTGGTCATGTACAGAATGTTAATGC
AGAGCTGGGTCAGGCTACGGTGTTCAACATATACAAGGTGAGAAAGCTCTGCCTCTGGT
TAGATTAAGGTATGTGGATTGTCCCGTGCATTCTGTCCATGTGTCTGGGAACCTCATGT
AATCATGAGAACTAAGGAATCCACCGATTCCTTAGACACCAGGTTTCTAAGGGCCTAGG
CATTGTGTCTGGTTTTGTTTTTATCTTCCCAAACATGGGTTTTTTCCAGCTTCACTTAA
TATGTCTAGCAGATAAAGCTATTTCAGTTAGGCACAGTTCCACTGCTCTTAGAAACATT
GAGGATGAATTTTCCTTGTTTGTACCTGCTTCTTGCCAGAGACTAAAAACTGATAATTT
GGCTGCTGGCAAGATTCCACTGCAACAGCTAGTAGAGTGTTCTGGAACGATGCCAACAG
CAGACAGATTGCATTAAATACTTAAACTACTATTTTAGATCAGTTCTGCAGTTGGGGAC
CTGAAATCAGCGGTGGATCCACTTTCTACTTTCCAGCTTACTTTGCTCTGGGTTTTTTA
AATGTCTTCTGTGATTTCCTCCAAATGGTTATCTCAAAGTGGCTTCTCTCTCTCTGTCT
CTCTCTCTAACATGÑ
Ä ë è í à ô ð à ã ì å í ò à Ä Í Ê 800-900/876 ï.í. (Õ-õðîìîñîìà, PIC «−»)
TTTATAGAGAGAGAGAGACAGAAAAATTCTGTAACTGTGTAACTGTTCTTATATTTAAT
CCATGTGACACATTTAATATTGATTTTCTCTGTCCTAGGATCTATGTGATGACTTATTT
GAAATAGCCAAGTGGATTTTAGGGTTATCACTAAAAACACACCATTGATTGGAGCAATG
CTGTCCAATTGAACTTTCCCAATGAGGGGAATGTTATATATCTGTGCTGGAGTAGCTAC
TAGCCAGATAGAGTTTTTGAGCAAATGACATGAAGCTAGTGTAGCCGAGGAACTGAATT
47
Ïðîäîëæåíèå òàáëèöû 2
TTTAATTTTATTTAATTTTACTTAGCTTATATTTAAATAAATGCATTTGGCTAGTGGTC
ACTCTGTTGGGCAGAGCAGCTCTGTCCAAAGATAAATTTTGAGAAAAAAATTGTTAAAG
AAGCCTAGTGTTTGTCCTTATTTTTAAATAAACTATCCTCTAAGCTCTCAGATTCATCA
CCTTCCATTAATTAAACAGAATACTATGAAATTCTAAAGGTTCTCAGAAGTGACCATTT
GATTTGTGAGACAAGGGTAAAGTGGAAAACAGGTGATTGGGAGTCTGGATTCTCCCCTG
CTTTCCCATCTCTCTACCTTCAACAAAACAGCATATTTGTATGTGTTTAGTAAATTAAT
TTTTCACTTAACTCTTTACTCAAATAAAGATTTTATTCCACTTAAAATCTTTTAGAAAC
CACTACCTTGAACAACAGATTTTTATCTATATTCAGGAGCAACATGCAATATCTACCCA
GTATGTGCCTGTGCATATTTATAGGAAGGGAAAGTCACTATCCATGTAGAATGATATGA
GAGGCATAAGAAGGTCTCTCCACCTCCAAGTGTCTCTCTCTCTCACTCAC
Ä ë è í à ô ð à ã ì å í ò à Ä Í Ê 800-900/871 ï.í. (18-ÿ õðîìîñîìà, PIC «−»)
AAGACAGAGAGAGAGAGACAGAGAGACAGAGAGAGAGAGAGAGAGGGAGAGAGACTGAC
TTAAAAGAGGCAGCCTGAACTGGATCTTGGGGAAGCAGCTCGGAATATATTATTGTACT
TGTTTTTTTGTAATACTAAGCCAAGCCAATAGATCAAAGGCAGCACAAAGGGAAAAGGC
AGGATAACGTAACGAACAGTTTCTGACTAAAATTCCTCTATCACTCCCCCATATGGCTC
GACACCTCCACCACAACAAGCAAAAGACACACACACACACACACACACACACGCACACA
CACGCACAAAACCTAGAGAGAGACAGAGAGACACAGAGAGAGAAGCATCACTTGTCTCT
AATCAAAATTCTCTCGTCTTTGTGTTTTCCTCCCTCCAGCACCCCACACTGCGCCCCCA
GCCGCCCCCTCCCCGCGCCGGGCAGCTCCGGGGCAGGCCGGGGGCGCGCGCGATTGTCT
TCTCTGGAAGAAAGTTGCTCCAAGTGGGCCCGGGCGCTCCCCGCGAGCCGAGGGCTGGG
CTTGCGGGAGGCCGGAGGCCGGGCTGGCGCCGCGTCCCCGCGGCCAGCTCGGGAGCGTC
AACGCGGCCCCGCGCCGGGTGATCGGGCTCAGAGCGAGGGGAACGAGAAGTTTCCTTTC
CCAGTCTGAGCTGGCGCTAGTTCGCTTGTTTGTTTTGGGGGGTTGGTTTTTCTTTTTAA
ACTCAGTATCCTCCGTCAGCTCCCCCTCGGCACCCTCTCTTGTCCCCAGCTCTCTCCCC
TCCCCCAGGCGCCTTCTGCGCTCTTGCCAATCACTTTTCTCTTTTATTCCCACGATTTT
GTTTGGGGGTAATACCTAGGGGGTCGGTCTCTCTTTCTCTCTCTC
Ä ë è í à ô ð à ã ì å í ò à Ä Í Ê 800-900/857 ï.í. (3-ÿ õðîìîñîìà, PIC «−»)
CTGTTAAGAGAGGAGAGACAATTTGATGTGCAGCTTCTGACTGTCTCAGGCTACTGATA
AGTACAGTTGATGGCAGCATTAGACTCTGTTTTAGTTATCTATTGCCCAACACTTTAAA
AAATGATCTTTTTCCTCATGATTCTGTGGGTTGACTAGGCTGAGTTGGGTGGTTTTTCG
GCCCCTTATGGTGTCAGCTAGGGTTACTCAGTTGGTTGCATTCAGTTAGCATCTTGGTT
GGCTAGAAGTTCCAACAAAGCTTCACACAAATGCCTCACCCTTTGCTGGTTCTCCACGT
ATCCTTTTTACGTGGCTAGCTTGGGCCTCCTTTCAGTATGGTGGCTTCAGGCTATTCAA
ACTTCTTACCCTGCCACTGGCTTCAAGGGGGAAAACAGCAGCTGCTAAGCCTCTTAAAG
CCTAGAACTGCATCTGGCACAGTGTCACTTCCACCTAAATTATGAGAACAACCCAAATT
CAAGGGAAGGGAGGTAGACGCCACTTCTCTATGAGGAGAGTGGCTTGCTGATAGAGTGA
GGGAAAGAATTGATGGCAGCCATCTTTGGAGAATCACCACCACAGCCTCATTACTCACT
TTACATTAGCACTATTTAATTTTATGTTAGATTTTCTTATGTATCTTCAGCTCCCTTTA
TAAGAACAATCTTACATTCACTTACCAGTTCCAGCCAGTGTTTTCTGGTCCTTTAGGAA
AAGTGGTCCTAATTATTCTTAATAGTTGGTACTATATAGTTAGGTCTCACTTATGTGCT
CAGTACTGAGTTTAGTTCTTTATGTATATGAGCAAGTCCTTACAATAACCCTGTGAGGG
AGATATTATTATGTCTCTCTCTCTATTTTTT
Ä ë è í à ô ð à ã ì å í ò à Ä Í Ê 800-900/832 ï.í. (6-ÿ õðîìîñîìà, PIC «−»)
GGTGAAGAGGAAGAGAGACAGTGGGCTGAGATCCTACGCCTGAAACTTATCTGGACAGA
AAGGATGCCTTTAATCAGGCCTGACTAACTGTCTGCACCCCCACATACACACCTTCACA
CAAATTCTTTGGAGGGCAAGGGGGGAAGAGGTGTAAAGGCCAACGCAGACGGCAGTTTC
ATCCTAGGATAAGAAGAGCCATCACAACATTCAAGAAATGTTAGCATATTTTCTTTTCC
TGGAAGTGAGGACCAGGGCACCAAAACAGTGACAGGCACATTGTGCCTCGAACTGCTGG
AGGAGGCAGTGCATTTTAGACAAGATGTTTAAGCTCCCTGCTTCCATGTTTTCATCTGC
AAATCAGCAGTTACGACAGTAACCACGCCATAGGGTTATGATGAATTTTAATGGGATAA
TCCAAGTCAAGCACTTAGCACGTGCCTGGCACAAAGGTGACGCTCAATAAAAGTGAGCT
CTTGTCATTCTTTGGAGCACCCCGTACATTCTGTCTACCCCCTGCTCACCCTGACCCAA
GGCGTCTGGTTTAATTTAGTGTCCAAAACCAGCCTAGGCAGATGACCTGGAAGCCTCAA
AGTCTGAACACCTGGGGCTTACAGGGGTGACTGGAGGACCACCCGGATCTCCTCACCTG
AGGATCCATCTTTGTGAGCGCCCACCCAGCTAGCCTGGGGTTCTGTCCGGGTCCTCAAG
TGGGAGACAGCTATGCTGGAGTGAGGCTGACCCTGGACCTGGGGTTAGGATTTTCAGGA
TTGGAACCCTAACTCTGCCATTTACCAACTTGGTTACCTTGAGCGAGTCTCTCTCTCTC
TAGCAT
Ä ë è í à ô ð à ã ì å í ò à Ä Í Ê 800-900/823 ï.í. (19-ÿ õðîìîñîìà, PIC «−»)
TAGAGAGAGAGAGAGAGACAGATGGGGGGAGGGTGTGGGGGAGGGCGAGATTCATTGAT
TGATTGCAAGGAATTGTAATGCAATTGTGAGAGCTGTCTGGGCATGTCCCAAATCAGGC
CATCAGGGAGGGCAGTTTGGAAACTCTTGGTCAGGAGCAGATGCAGGAGTCCACAGGAA
ATTTCTTCTTCCTCAGGGAAACCTCAGTTCTGTTCTGAAGGCCTTCAGCTGAATGGTTC
AGGCTCACTGAGATTATCCAAGATAGTCTCTTATACAGCCAACTGATCCCATCACTGAA
ATACCTTCATAGCAACACCTGGAGCACAACAACCTAGAGTGCTGTTCGATTGCATAATA
GAGTACCATAGCTTAGCCAAGATGACACAAAACTAACCATCACAATAAGTACAAGGGAA
GGGGCAAGACGCTACTACAGTATTCCTTGGACTAGGTGAATGGGGGCAATAATATTGGG
TGTATTGTTCGGTAGTGGCCGGCTGACTGGTCCCTGTTTGGGAGGCGGAGTGTGCACAA
GGTTCGCATGCTGTTGTGTCACAGCCCAGGACCCTTGGTGTACTTTTTTCTATCCCAGC
CCTAATATAACCTTCTGTCTTCCAGTGTATAGCATGAAGCTCTAGCTTCATTCTTCCAA
TTCAATCCCGTGTTCTCTGAAGAAAAAATGCCCAAAGCAATTTTCACCCCATCACACAC
TAGTAATCATTTTATTTTTCTGATTATGTTTCTTTCTCTCTTTCAGCCCTTTGGTAAAT
TACGCAGGGACCCTTTATTCTCATTCTCTCTTTTTCTGTCTCTCTGTGTCTCTATG
Ä ë è í à ô ð à ã ì å í ò à Ä Í Ê 7401/739 ï.í. (16-ÿ õðîìîñîìà, PIC = 0,29)
GACAGAGACAGGGAGAGACAGACAGACAGACAGAGACAGAGAGAGAGAGAGAGAAAGTG
AGCCAGACGAAGCTGTATCCCTTTTCATGAGCTAGCATCAGAAAATAAGTCACAAAGAC
TAAGACTAGCCCTTATTCAATAGTGAAGGCTTAGATGCGACCATTTGATGAGAATGTCA
AGTAATTTTCAGACATGTTTTAAAACCATCACAGTTGTATATGTTCTTCCAGGATGTTG
ATCAACTCCATTTCTTTAACATCTCCTCTCATATCTAGCCTCTCCTCCCCAGCCTATAG
TTTATTCCTCAGTACGTGTAATGGTTCATATTATGTACCAACTTGACTAGGTTACAAGA
48
Ïðîäîëæåíèå òàáëèöû 2
TGCTCATATATTTGGTTAAACAGTATTTCTGGGTGCATCCCTGAGAGTGTCTCCAGAAG
ATTAGCATTTAAGTCTGACTGAGTAAAGAAGATGTCTCCCTCGTCCCCCAGTTGGGGTG
GGCATTATCCGATCTATGAGGGCCTCAACAGAACAAAAAGGTGGAGGGAGGGAGAATTT
GCTTTCTCTGCTTAAATGCTTGAACTGGGACATTGGCTTTTTTCCTACCAGCTCTCCTG
GTTCTCAGCCTTCAGGCTCAGCTAGAATGACACCGCTAGCTTTTATGCGTCTCCAGCTT
GTAGATGGCAGATTGTGGGACTTCTCAGCCTCCATAATTGCATGAGCCAATTCCATATA
ACCTCTGTCTCTGTCTCTCTGTCTCTGTCTG
Ä ë è í à ô ð à ã ì å í ò à Ä Í Ê 580/585 ï.í. (1-ÿ õðîìîñîìà, PIC = 0)
CGCTGAGAGAGAGAGAGACCAAATGCCCACAGACGGCTCATCCTAACTCGACATGGAGG
AAAAGTCCTACTGAGTGCTAAAAGTAATTCCCAGAGTATGGAATTCTGAATGACGGGCT
GAGGTAATGATGGGCACCTTCAGGAGGCTGGCACCCCCATCTCAATTATACCAACCTCA
ATCTGCTTATACCTGTCAGTTTGTAAAAGAACTCTCCTCTTCCTGTCTTAGCCAGGCTG
CTCATAAGAGTTCCAACAAACAACCAATAATTAATAGAAGGTTGGTCCTAGGTTGGTCT
TAACCAAGCCATGAAGGATGTGTGTGTAGATTTTATACTCCAGCAGAGGGCAGTACCAC
ATAAAAATTACTATTAAGGAAAAACTGAGGTGCAGCCTCTTTTGTCAATTACCCTGAAT
TTTCTGTATTATTGAATAATTATTATTTACTGTCACAAAAACGGGTAACTTGTTCATAC
GGTTATCTTGAGTATCAATGGACAATCCTGGCAATCAATTTAAGCCCTAACAATTTTAA
ATCTTACAATTTTACATAATACTTGCTTTACGTGAGTCTCTCTCCTCTCACATG
Ä ë è í à ô ð à ã ì å í ò à Ä Í Ê 550/637 ï.í. (Õ-õðîìîñîìà, PIC = 0,29)
CAAAACGAGAGAGAGAGACCCAGACTTCTCATAGAACCCGAATTAGGACCGATACACTC
CTTACTGCCAGAATGTAAGAGGAATTTCCCCTATAAGTCCCGTGAGTCAGAACTCAGGA
AAATAATGAGGGGCTTTCCATGGCTCTCCTGAGGAAAAGAGAAATATCTATTTACTTGA
AGAACATGGAATGTGTTTCATACCACTTTGTCTTTCTCTAGGACTGAGGTCTGTTGGCT
TTTTAAAATATAGCTACCCAAGGAGTGGGATCTCTCTCTTTCTCTCTCTCCATGTTTTT
CTCTTTCTCCATCTCTCTCCATCTCTCTCCATCTATCTCTCCATCTCTCTCCGTCTCTC
TCTCCATCTTTTTCTCTTTCTCCATCTCTCCATCTCTCTCCATCTGTCTCTCCATCTCT
CTCCATCTCTCTCCATCTGTCTCTCCATCTCTCTCCATCTCTCTCCATCTCTGTCTATC
TCTCTCCATCTTTTTCTCTTTCTCCATCTCTCTGTCCATCTCTGTCCATCTCTCTCCGT
CTGTCTCTCTATCTCTCTCCATCTCTGTCCATCTCTCTCCATCTCTCTCTCCATCTATC
CTCCATTTCTCTCCATCTCTCTCTCCATGTCTCTCCATCTCTCTCTC
Ä ë è í à ô ð à ã ì å í ò à Ä Í Ê 550/572 ï.í. (10-ÿ õðîìîñîìà, PIC = 0,29)
AAGAAAGAGGGAGAGAGACAGAGAAATACTCTGCTCTCTCCAGCACTCCAGTCTTCTGC
CAGTGCCTCCCATTGGCCAGACCTACCTGGAAGGCTGAGGGCAACAGACTCTGGGAGCT
GTAGTTCCCGGAAATACCGAGAACAGTAGGAGGTGGGGGTAGGAGGGATACAAAAGGGG
GGCAAAAAGCATCTGGCATTGATTCTAAAACTAACAGGAAAAACTAGGCTGTGAAAAGT
CAAAAGCTCTTAGGAAAATGTCACTTTGGAGATGAGCTCACATCTCTTATGAAGTTTAG
CACAGCCAGTTTTACCTCTGGTGTTGGAACAGAAAATTCTCCTTTTGATTGGGCAATAG
ATGACATCTTTGGCTTTGCTCTGGGGTCATCTTGTACGAACAATATGAATATTTCAATG
CTAAAATCATTCAGCTCAGTGACATGCTCATAACTTGAGAAGTTGCCAGAAACTGAGAA
GAAGAACCAGGGTAAAAGCAGTTTCACACCTCCCATTTCGTCCTTACTCAGAAATCTCA
GTCTCTCTCTGTTTCTATCTCTGTCTCTCTCTCTTTCTCTT
Ä ë è í à ô ð à ã ì å í ò à Ä Í Ê 500/494 ï.í. (1-ÿ õðîìîñîìà, PIC = 0,44)
CACAGAGGAGGAGAGAGACTGTTCCCCAGAACAGAACTGGGTGCAGAACCAGAAGAAAG
TGAGACTCCTGCTGGGGCCAAAAACGTCAATGAGTTCTGATGTAGTGGACAACGACACC
ACTGACAGAGCGGTTGTAAGGAGAAAAGGGACCAAACAGTAAAGCACTGGGCACAGTGC
CCTGTCCCTTCTCAGGGGTCAGCAGCGTTAGCTGTGCTAACCATCGCTTCTCTCAATCC
TCAGCAACTCTGTGAAGCATCTCCCCCATTCCCCAGATGGGCGACCGGGGCTCGAGCTG
GTGATGGGGTTGCCTGAGGCTGCCTAAGTGCCAGGCAAGTCTCTGATCCAGCCCGTCCG
CAGGGCCGGCTACATACACTGCAAAGCCTTGTGCAAACGAAAGCAAGGAAGCCCTTGTT
CAAAAAGCAGGAAAAATACCATTAAGAGAGCTAAAATATAAAGCTTTTTCCTTTCTTCC
AGTGTCTCTCTCTCTCTCGCCA
Ä ë è í à ô ð à ã ì å í ò à Ä Í Ê 490/489 ï.í. (28-ÿ õðîìîñîìà, PIC = 0)
GAGAGAAAGAGAGAGAGACCACAATGAGATATAAAATACAGGACATAGTTTAAATTACT
GACAAAGTGGCAAACTGGTGTGTGTGTCTTGTGGTACCCTTCGGGAAGCTGTCCTGTCT
TTTTTTTTTCATATCACCTGATACCTCCAGCTCCACTCGGCTCAAGGTTTGTCAGGTCA
AGAGCTCAGAAGAGCAGCCGCACACAGCGCTCTCCACAATCTCTCGCCTGTGCTCGCCC
AGATAGACCTCAGGCCCTGGCCCTTTCATGTCCTCTCCCAGCTCTCAGCCTGGCTCCCC
CCTCAGAAAGCCAGAGTCCCAGCCGAATCAGAGGAGCTGGTGTTAGAGTCCCCGAAGAG
GTCACCTCGGCCGTCCTGGGGGCACAGCTGTCATGGGTTTCAGAGCAGCAGGTAAAATG
GACTGCAAGTTGAGAGCAGACCCAACAGGGGAGGAGTCAGACAGGCGCCCACACACCGT
CTCTCTCTCTCACACAC
Ä ë è í à ô ð à ã ì å í ò à Ä Í Ê 380/378 ï.í. (11-ÿ õðîìîñîìà, PIC = 0)
ACACACAGAGGAGAGAGACGTAGGGAGAAGAGAAGGCCGTGTGAAGACGGAGGCAGAGA
TTGGAATGATGCAGCCACAAGCCAAGGAGCATCTGGAACCATCCGGAAGCTACAAGAGG
CAAGCAAGAATTCTCCCCTACAGCCTTTGGAGGGAACGTGGCTCTGCCAACATCTTGAT
TTCAGACTTCTGGCCGCCAGAACTGTGAGAGAATGAATTTCTGTTGTTTATAGCCACCA
AGTTTGTAGTCATTTGTTATGGCAGCCTGAGCAAAGTAACACAGGAAATTACTGTGATA
ATGTAGCAAAACGTTTAAAAGCCTGTTTCCCCAAGCACGTCTCTCCTTCTCTGTCTCGC
TGCGTGTCTCTCTCTGTCTCTCTC
Ä ë è í à ô ð à ã ì å í ò à Ä Í Ê 380/377 ï.í. (21-ÿ õðîìîñîìà, PIC = 0)
TGAGGAGAGGGAGAGAGACAGGGGAACACTGGTTGGTGGAGCAGTCATAACACACACAT
TTATCAATTGTTAGCCATCTTATATGGTTGAGGTTCATCGCCCCTCAAAACAATTACAA
TAGTGCCATCAAAGATCACTGACACAGATCACCACTACAAATATAATAATAATTAAAAA
TTTGAAATTTTGTGAAAATTACCAAAATGTGACACATAAAAGTGACCAAATGCTGCTGG
GAAAATGACTCCACTAGACTTTCTTATTGCAGGGTTGCCACAAACCTTCAATTTGTAAA
AATGCAATATCTGCAAAGTGCAATAAAGTGGGCTGCGAAAAAATGAGTTAGGCTTCTCT
CTCAGTCTCTCTATCTCTCTCTA
Ä ë è í à ô ð à ã ì å í ò à Ä Í Ê 360 (370)1/364 ï.í. (5-ÿ õðîìîñîìà, PIC = 0,421)
GAAAAAGAGGAAGAGAGACCGGAGCTCTTTCTTTCTCTCTTTCGATCATGTGAGGATAC
49
Ïðîäîëæåíèå òàáëèöû 2
AGCAAGACTGTCTGGGAACCAGGAAGAAGGCCCTTACTGAGAACCTAATTGGCAGGCAC
CTTGCTCTTGGACTTCCCCATCTCTTATTCTGTTGTTTAAGCCACTCAGTCTATGGTAT
TTGTTATAGCAGCCCATCTTAGTCATAAGACATACATATATATACACATATACGTATAT
GTGAGTCTGTGTGTGTGTATATATATATGTATTTTTTTCCATTTCCTAAATTCATGCAC
TTTCAAAAAATGCCTTCTCTTCCTTTTTCACATTCTGTAGAAATAGTACAGTCTCTCTT
CTCTTCCCCT
Ä ë è í à ô ð à ã ì å í ò à Ä Í Ê 310/309 ï.í. (5-ÿ õðîìîñîìà, PIC «−»)
TTCACAGAGAGAGAGAGACAGAGACAGAGATGAGAGAGAGAGGACAGGAAATGCAAAGA
GATAACTCTAAATAGAGAGGCAAAAGGGAAGAAAATAAGGAACATATAGATAGAATAAT
ATTATCATTCCAGAGACTCTCAGTGAATTTCTGTAAAGTCCATAGGGACAGGGCCCCTT
ATCGCCATGTTTCAGGGATTTGATTAAGGATGCTCAGCAGGCACTTGACAGAGTGAGCT
TGTTCAGTTCTAATCCTTCGAGGAACCATTACTCATTCTGCAACATTGAGCCATGTCTC
TCTTTCCCTCTTCT
Ï ð è ì å ÷ à í è å. Ïðåäñòàâëåíû ðåçóëüòàòû àíàëèçà ñèêâåíñîâ èç Gån-Bank (http://www.ncbi.nlm.nih.gov)
äëÿ ïîñëåäîâàòåëüíîñòåé ðàçìåðîì äî 2000 ï.í. íà ñîîòâåòñòâèå ïîëó÷åííûì ñîáñòâåííûì ýêñïåðèìåíòàëüíûì äàííûì ïî ISSR-PCR àìïëèôèêàöèè ôðàãìåíòîâ ÄÍÊ ñ ïðàéìåðîì (GA)9Ñ (ôàêòè÷åñêàÿ äëèíà/ðàñ÷åòíàÿ äëèíà). PIC — èíäåêñ ïîëèìîðôíîãî èíôîðìàöèîííîãî ñîäåðæàíèÿ ëîêóñà, PIC «−» îçíà÷àåò, ÷òî â ýêñïåðèìåíòå íåâîçìîæíî îöåíèòü ïîëèìîðôèçì ôðàãìåíòà â ñâÿçè ñ îòñóòñòâèåì ÷åòêîãî
ñîâïàäåíèÿ ýêñïåðèìåíòàëüíûõ è ðàñ÷åòíûõ äëèí ôðàãìåíòîâ, 1 (â âåðõíåì èíäåêñå) — îïóáëèêîâàííûå
äàííûå (6). Ñâåòëî-ñåðûì âûäåëåíû ñàéòû îòæèãà ïðàéìåðà, ñåðûì — òî÷êè îøèáêè îòæèãà; ïîñëåäîâàòåëüíîñòè, ïðåäðàñïîëîæåííûå ê ôîðìèðîâàíèþ G4 êâàäðóïëåêñîâ, îòãðàíè÷åíû ðàìêàìè.
Äëÿ êëàñòåðà 800900 ï.í. òàêæå áûëè íàéäåíû ïîòåíöèàëüíî àìïëèôèöèðóåìûå ó÷àñòêè ÄÍÊ èç
ðàçíûõ õðîìîñîì: 900 ï.í.
Êëàññ
×èñëî Îò îáùåãî ÷èñëà ïîâòîðîâ, % (24-ÿ õðîìîñîìà), 876 ï.í. (ÕERV/ERV3
47
15,93
õðîìîñîìà), 871 ï.í. (18-ÿ
DNA/hAT
32
10,85
õðîìîñîìà), 857 ï.í. (3-ÿ õðîNonLTR/SINE/SINE2
30
10,17
ìîñîìà), 832 ï.í. (6-ÿ õðîNonLTR/L1
29
9,83
LTR/Gypsy
28
9,49
ìîñîìà), 829 ï.í. (19-ÿ õðîNonLTR/CR1
17
5,76
ìîñîìà). Íàèáîëüøóþ òî÷DNA
15
5,08
DNA/EnSpm/CACTA
13
4,41
íîñòü îòæèãà èìåë ôðàãìåíò
DNA/Mariner
10
3,39
äëèíîé 871 ï.í. èç 18-é õðîLTR/Copia
10
3,39
ERV/ERV1
9
3,05
ìîñîìû (ñì. òàáë. 2). ÎòìåERV/ERV2
7
2,37
òèì, ÷òî ïðè ïîâûøåíèè
DNA/Ginger2
5
1,69
DNA/Kolobok
5
1,69
òåìïåðàòóðû îòæèãà ïðèìåðLTR/BEL
4
1,36
íî òàêîìó ðàçìåðó ôðàãìåíDNA/Dada
3
1,02
òà ñîîòâåòñòâîâàëà çîíà, êîDNA/Harbinger
3
1,02
Interspersed_Repeat
3
1,02
òîðàÿ ÷åò÷å âèçóàëèçèðîâàDNA/Helitron
2
0,68
ëàñü â âèäå êëàñòåðà. Äàëåå
DNA/MuDR
2
0,68
DNA/Polinton
2
0,68
íà ýëåêòðîôîðåãðàììå ðàñLTR
2
0,68
ïîëàãàëñÿ ôðàãìåíò ~ 580 ï.í.
NonLTR/L2
2
0,68
NonLTR/Tad1
2
0,68
Ñîãëàñíî ðàñ÷åòàì, îí ìîã
Simple/Sat
2
0,68
ñîîòâåòñòâîâàòü ó÷àñòêó äëèDNA/Novosib
1
0,34
DNA/piggyBac
1
0,34
íîé 585 ï.í., ëîêàëèçîâàíDNA/Transib
1
0,34
íîìó â 1-é õðîìîñîìå (ñì.
DNA/Zator
1
0,34
òàáë. 2).
LTR/DIRS
1
0,34
NonLTR/CRE
1
0,34
Îñîáûé èíòåðåñ ïðåNonLTR/I
1
0,34
äñòàâëÿëà
çîíà, ñîîòâåòñòNonLTR/Rex1
1
0,34
NonLTR/RTE
1
0,34
âóþùàÿ 550 ï.í. Èñõîäÿ èç
NonLTR/RTEX
1
0,34
íàéäåííûõ íóêëåîòèäíûõ ïîNonLTR/Tx1
1
0,34
ñëåäîâàòåëüíîñòåé, îíà, âåðîÿòíî, ãåòåðîãåííà è âêëþ÷àåò ó÷àñòêè èç 10-é õðîìîñîìû (ïîëíàÿ äëèíà ôðàãìåíòà 572 ï.í.) è Õ-õðîìîñîìû (ïîëíàÿ äëèíà 637 ï.í.).  êàæäîì èç
íèõ íàõîäèëèñü òðè ñàéòà îòæèãà ïðàéìåðà — ïî îäíîìó ïðÿìîìó è ïî äâà
îáðàòíûõ. Òàêèì îáðàçîì, ñóùåñòâóåò âîçìîæíîñòü àìïëèôèêàöèè êàê ïîëíûõ ôðàãìåíòîâ (ñîîòâåòñòâåííî 572 è 637 ï.í.), òàê è èõ êîðîòêèõ âàðèàí3. Ïðåäñòàâëåííîñòü äèñïåðãèðîâàííûõ ïîâòîðîâ ðàçëè÷íûõ êëàññîâ â ñåêâåíèðîâàííûõ
ó÷àñòêàõ ãåíîìà ó äîìàøíåé ëîøàäè Equus
caballus, ôëàíêèðîâàííûõ èíâåðòèðîâàííûì
ïîâòîðîì (GA)9C
50
òîâ (550 è 552 ï.í.) (ñì. òàáë. 2). Íå èñêëþ÷åíî, ÷òî ïî óêàçàííîé ïðè÷èíå ýòîò ó÷àñòîê îòíîñèëñÿ ê ÷èñëó ëîêóñîâ, êîòîðûå âîñïðîèçâîäèëèñü
íàèáîëåå ñòàáèëüíî, õîòÿ îí è íå áûë îäíèì èç ñàìûõ ÿðêîîêðàøåííûõ.
Ìàæîðíûé ëîêóñ äëèíîé îêîëî 490 ï.í., ïî-âèäèìîìó, âêëþ÷àë
ôðàãìåíò ÄÍÊ èç 28-é õðîìîñîìû, èìåþùèé äëèíó 489 ï.í. è âûñîêóþ
êîìïëåìåíòàðíîñòü ôëàíãîâ ê ïðàéìåðó (GA)9C. Ìàæîðíàÿ çîíà ~ 380 ï.í.
ìîãëà ñîñòîÿòü èç íåñêîëüêèõ ôðàãìåíòîâ: 377 ï.í. (21-ÿ õðîìîñîìà), ó êîòîðîãî ïîñëåäîâàòåëüíîñòè ôëàíãîâ íàèáîëåå ïîëíî êîìïëåìåíòàðíû ïðàéìåðó (GA)9C, è 378 ï.í. (11-ÿ õðîìîñîìà).
Òàêèì îáðàçîì, â õðîìîñîìàõ äîìàøíåé ëîøàäè áûëî íàéäåíî 99
ôðàãìåíòîâ ÄÍÊ, êîòîðûå ïîòåíöèàëüíî ìîãóò áûòü àìïëèôèöèðîâàíû â
ÏÖÐ ñ ïðàéìåðîì (GA)9C (ôðàãìåíòû ñ íàèáîëåå òî÷íûìè ñàéòàìè îòæèãà íà ôëàíãàõ — ñì. òàáë. 2).
Àíàëèç íóêëåîòèäíûõ ïîñëåäîâàòåëüíîñòåé â ðàñ÷åòíûõ
ñ ï å ê ò ð à õ ô ð à ã ì å í ò î â Ä Í Ê Ô È Ï (GA)9C. Ìû ïðîàíàëèçèðîâàëè 99
ó÷àñòêîâ ãåíîìíîé ÄÍÊ ëîøàäè, ôëàíêèðîâàííûõ èíâåðòèðîâàííûì
ïîâòîðîì (GA)9C è ïîòåíöèàëüíî ñïîñîáíûõ ê ôîðìèðîâàíèþ ïðîäóêòîâ àìïëèôèêàöèè â ÏÖÐ ñ ýòèì ìèêðîñàòåëëèòîì â êà÷åñòâå ïðàéìåðà, íà íàëè÷èå ôðàãìåíòîâ ìîáèëüíûõ ãåíåòè÷åñêèõ ýëåìåíòîâ (ÌÃÝ)
ðàçíûõ êëàññîâ ñ ïîìîùüþ áàçû äàííûõ http://www.girinst.org (òàáë. 3).
Îêàçàëîñü, ÷òî íàèáîëüøåå ðàñïðîñòðàíåíèå èìåþò ó÷àñòêè ãîìîëîãèè
ñ ýíäîãåííûìè ðåòðîâèðóñàìè III êëàññà, êîòîðûå ñîñòàâëÿëè ïðèáëèçèòåëüíî 15,93 % îò îáùåãî ÷èñëà âñåõ âûÿâëåííûõ ÌÃÝ. Íà äîëþ ãîìîëîãèè ê ýíäîãåííûì ðåòðîâèðóñàì I è II êëàññîâ ïðèõîäèëîñü ñîîòâåòñòâåííî
3,05 è 2,37 %. Çíà÷èòåëüíî ðàñïðîñòðàíåííûìè áûëè ó÷àñòêè ãîìîëîãèè ê
DNA/hAT (10,85 %), NonLTR/SINE/SINE2 (10,17 %), NonLTR/L1 (9,83 %)
è LTR/Gypsy (9,49 %).
Îöåíèâàÿ ñïåöèôè÷íîñòü ñâÿçè ó÷àñòêîâ ãîìîëîãèè âî ôðàãìåíòàõ
ÄÍÊ, ôëàíêèðîâàííûõ èíâåðòèðîâàííûì ïîâòîðîì (GA)9C, èìåííî ñ ýòèìè ÌÃÝ, ìû âûïîëíèëè ïîèñê ó÷àñòêîâ ÄÍÊ ñ ïðÿìûìè ïîâòîðàìè íà
ôëàíãàõ. Îêàçàëîñü, ÷òî òàêèõ ó÷àñòêîâ â ñåêâåíèðîâàííîì ãåíîìå ëîøàäè
ñóùåñòâåííî áîëüøå, ÷åì ôëàíêèðîâàííûõ èíâåðòèðîâàííûì ïîâòîðîì
(GA)9C (99 ôðàãìåíòîâ).
4. ×àñòîòà ìîáèëüíûõ ãåíåòè÷åñêèõ ýëåìåíòîâ (×ÌÃÝ) è äîëÿ ó÷àñòêîâ ãîìîëîãèè ê íèì (ÄÓÃ) âî ôðàãìåíòàõ ÄÍÊ, ôëàíêèðîâàííûõ èíâåðòèðîâàííûìè
è ïðÿìûìè ïîâòîðàìè ìèêðîñàòåëëèòà (GA)9Ñ, ïî ñðàâíåíèþ ñ äîëåé íóêëåîòèäîâ ÌÃÝ â ñåêâåíèðîâàííîì ãåíîìå (ÄÌÃÝÃ) ó äîìàøíåé ëîøàäè
Equus caballus
Ãðóïïà ÌÃÝ
ÄÌÃÝÃ, %
Èíâåðòèðîâàííûå ïîâòîðû
×ÌÃÝ, %
ÄÓÃ, %
Ïðÿìûå ïîâòîðû
×ÌÃÝ, %
ÄÓÃ, %
LINE_L1
16,25
10,31
4,83
11,56
9,29
LINE_ CR1
0,26
5,84
4,70
6,80
2,85
SINE
2,85
9,97
4,45
13,61
5,63
LTR/Gypsy
–
9,62
5,22
16,33
5,42
LTR_ERV, III êëàññ
1,82
15,81
9,55
7,48
4,17
LTR_ERV, I êëàññ
1,53
3,09
0,96
8,16
4,34
LTR_ERV, II êëàññ
0,07
2,06
0,64
1,36
0,56
1
DNA/hAT
1,55
11,34
5,94
3,40
0,90
Ï ð è ì å ÷ à í è å. Ïðî÷åðê îçíà÷àåò îòñóòñòâèå ãîìîëîãèè â ñåêâåíèðîâàííîì ãåíîìå êðóïíîãî ðîãàòîãî
ñêîòà; 1 (â âåðõíåì èíäåêñå) — ïðèâåäåíû äàííûå ïî ãåíîìó ÷åëîâåêà (P. Arensburger ñ ñîàâò. Genetics,
2011, 188: 45-57).
 ñåêâåíèðîâàííîé ÄÍÊ ëîøàäè ñïåêòð ó÷àñòêîâ ñ ïðÿìûìè ïîâòîðàìè (GA)9C, èìåþùèõ ãîìîëîãèþ ê ðàçëè÷íûì òèïàì ÌÃÝ, ñóùåñòâåííî îòëè÷àëñÿ îò ïðåäñòàâëåííîãî âûøå (ñì. òàáë. 3) ïî ÷àñòîòå îáëàñòåé
ãîìîëîãèè ê ýíäîãåííûì ðåòðîâèðóñàì III êëàññà è DNA/hAT (òàáë. 4).
51
Èíòåðåñíî îòìåòèòü, ÷òî ïî íàøèì äàííûì äîëÿ íóêëåîòèäîâ, ïðèõîäÿùèõñÿ íà îáëàñòè ãîìîëîãèè ê ýíäîãåííûì ðåòðîâèðóñàì è îñîáåííî ê
ÄÍÊ òðàíñïîçîíîâ, âûøå ïîëó÷åííîé äëÿ ñåêâåíèðîâàííîãî ãåíîìà äîìàøíåé ëîøàäè (5) (ñì. òàáë. 4). Òàêèì îáðàçîì, ìîæíî îæèäàòü, ÷òî èíâåðòèðîâàííûå ïîâòîðû ìèêðîñàòåëëèòà (GA)9Ñ íàèáîëåå òåñíî àññîöèèðîâàíû ñ ó÷àñòêàìè ãîìîëîãèè ê ýíäîãåííûì ðåòðîâèðóñàì III êëàññà,
LINE_L1 è DNA/hAT, ïîñêîëüêó â ýòèõ ó÷àñòêàõ èõ äîëÿ âûøå, ÷åì â ñåêâåíèðîâàííîì ãåíîìå êðóïíîãî ðîãàòîãî ñêîòà.
5. Ïðåäïîëàãàåìûå ðåêîìáèíàöèîííûå õàðàêòåðèñòèêè ìîáèëüíûõ ãåíåòè÷åñêèõ ýëåìåíòîâ (ÌÃÝ) â ñåêâåíèðîâàííûõ ïîñëåäîâàòåëüíîñòÿõ ãåíîìà äîìàøíåé ëîøàäè Equus caballus, ñîîòâåòñòâóþùèõ ïîëèìîðôíûì è êîíñåðâàòèâíûì ôðàãìåíòàì ÄÍÊ â ISSR-PCR
×àñòîòà îáðàçîâàíèÿ
×èñëî, ðàçìåð,
Äëèíà
ëîêàëèçàöèÿ ó÷à- Ïîòåíöèàëüíûå ó÷àñòêè ðåêîìáèíàöèé êâàäðóïëåêñîâ (íå ïåPIC
ôðàãìåíòà
ñòêà, ñîâïàäàþ- ñ ÌÃÝ/ïîâòîðû/êëàññ
ðåêðûâàþùèåñÿ/ïåðåùåãî ïî äëèíå
êðûâàþùèåñÿ)
Ïîëèìîðôíûå ëîêóñû
1 (986 ï.í.,
DNA transposon/hAT/MER103C
23-ÿ õðîìîñîìà)
980 ï.í.
0,49
7401 ï.í.
0,291 1 (739 ï.í,
Non-LTR Retrotransposon/L1/L1-8_GM
16-ÿ õðîìîñîìà) Endogenous Retrovirus/ERV3/MLT1G2
Endogenous Retrovirus/ERV3/MLT1F1
Endogenous Retrovirus/ERV3/MLT2B2
637 ï.í.:
0,29 2 (637 ï.í.,
LTR Retrotransposon/Gypsy/Gypsy-53_PM-LTR
Õ-õðîìîñîìà;
Non-LTR Retrotransposon/L1/L1M6_5end
572 ï.í.,
10-ÿ õðîìîñîìà)
572 ï.í.:
DNA transposon/Mariner/Tc1/MamRep137
Endogenous Retrovirus/ERV3/LTR33A
Endogenous Retrovirus/ERV3/LTR33
0,44 1 (494 ï.í.,
DNA transposon/hAT/MER45C
1-ÿ õðîìîñîìà)
550 ï.í.
500 ï.í.
Êîíñåðâàòèâíûå ëîêóñû
1 (900 ï.í.,
DNA transposon/hAT/Homo5
24-ÿ õðîìîñîìà) DNA transposon/Dada/Dada-1_CSa)
1 (585 ï.í.,
0
1-ÿ õðîìîñîìà)
900 ï.í.
0
580 ï.í.
0
490 ï.í.
0
1 (489 ï.í.,
0
28-ÿ õðîìîñîìà)
380 ï.í.
0
377 ï.í.:
2 (377 ï.í.,
21-ÿ õðîìîñîìà; DNA transposon/Tigger1B_Crp
378 ï.í.,
11-ÿ õðîìîñîìà)
3 (G-Score = 11, 13, 14)/
14 (G-Score = 8, 9, 11, 13,
13, 7, 7, 7, 6, 7, 14, 14, 10, 11)
2 (G-Score = 4, 20)/1
2 (G-Score = 3, 4, 2, 3, 4, 4,
5, 5, 20, 20, 19, 20)
1 (G-Score = 12)/
2 (G-Score = 11, 12)
2 (G-Score = 12, 34)/
438 (G-Score = 4815 —
ñóììàðíûé)
1 (G-Score = 19)/
46 (G-Score = 563 — ñóììàðíûé)
2 (G-Score = 16, 8)/
3 (G-Score = 16, 15, 8)
2 (G-Score = 18, 18)/
14 (G-Score = 17, 18, 16,
17, 13, 14, 17, 11, 18, 12, 4,
13, 14, 18)
2 (G-Score = 15, 12)/
10 (G-Score = 14, 15, 13, 8,
10, 9, 7, 9, 8, 12)
1 (G-Score = 17)/
33 (G-Score = 410 — ñóììàðíûé)
378 ï.í.:
Endogenous Retrovirus/ERV3/MLT1C1
1 (G-Score = 10)/
2 (G-Score = 10, 10)
Ï ð è ì å ÷ à í è å. PIC — èíäåêñ ïîëèìîðôíîãî èíôîðìàöèîííîãî ñîäåðæàíèÿ ëîêóñà, G-Score — áàëëû
âåðîÿòíîñòè ôîðìèðîâàíèÿ G4 (ðàññ÷èòàíû ïî ïðîãðàììå QGRS, öèôðàìè óêàçàíî ÷èñëî ïîòåíöèàëüíûõ
G4).  êà÷åñòâå ïðàéìåðà èñïîëüçîâàíà ïîñëåäîâàòåëüíîñòü (GA)9C; 1 (â âåðõíåì èíäåêñå) — äàííûå,
îïóáëèêîâàííûå ðàíåå (5).
Ìû äåòàëüíî ïðîàíàëèçèðîâàëè ïî ÷åòûðå ïðåäïîëàãàåìûå ïîñëåäîâàòåëüíîñòè äëÿ ïîëèìîðôíûõ è êîíñåðâàòèâíûõ ëîêóñîâ, èñõîäÿ èç
äàííûõ î ñåêâåíèðîâàííûõ ó÷àñòêàõ ãåíîìà ñ íàèáîëåå âûñîêîé òî÷íîñòüþ îòæèãà ïðàéìåðà, áëèçêèõ ïî äëèíå ñîîòâåòñòâóþùèì ôðàãìåíòàì
àìïëèôèêàöèè (ñì. òàáë. 2, 5).  ðåçóëüòàòå â ÷åòûðåõ ïîëèìîðôíûõ ëîêóñàõ âûÿâèëè 11 ó÷àñòêîâ ìîáèëüíûõ ýëåìåíòîâ, èç êîòîðûõ ê ðåòðîòðàíñïîçîíàì (ìîáèëüíûå ýëåìåíòû I êëàññà) îòíîñÿòñÿ 8, òî åñòü 73 % îò ÷èñëà îáëàñòåé ãîìîëîãèè ê ðàçíûì ÌÃÝ. Ïî÷òè ïîëîâèíà èç ýòèõ 11 ó÷à52
ñòêîâ (45 %) ïðèõîäèëàñü íà äîëþ ýíäîãåííîãî ðåòðîâèðóñà III êëàññà
ERV3, òîãäà êàê íà òðàíñïîçîíû II êëàññà (ÄÍÊ-òðàíñïîçîíû) — âñåãî
23 %. Â îòíîøåíèè êîíñåðâàòèâíûõ ôðàãìåíòîâ ÄÍÊ áûëè îáíàðóæåíû
òîëüêî ÷åòûðå ó÷àñòêà ãîìîëîãèè ñ ÌÃÝ, ïðè÷åì òðè èç íèõ — ñ ÄÍÊòðàíñïîçîíàìè (ÌÃÝ II êëàññà). Òî åñòü â ãåíîìå äîìàøíåé ëîøàäè â
ïîëèìîðôíûõ ISSR-PCR ìàðêåðàõ, âûÿâëÿåìûõ ñ (GA)9C â êà÷åñòâå
ïðàéìåðà, ïî ñðàâíåíèþ ñ êîíñåðâàòèâíûìè ñ îòíîñèòåëüíî ïîâûøåííîé
âåðîÿòíîñòüþ ìîãóò ïðèñóòñòâîâàòü ó÷àñòêè ãîìîëîãèè ê ERV III êëàññà.
Íàïîìíèì, ÷òî ýíäîãåííûå ðåòðîâèðóñû ìëåêîïèòàþùèõ ïîäðàçäåëÿþòñÿ íà òðè êëàññà â çàâèñèìîñòè îò èõ ïðîèñõîæäåíèÿ îò ýêçîãåííûõ ðåòðîâèðóñîâ, êîòîðîå îöåíèâàåòñÿ íà îñíîâàíèè ãîìîëîãèè ïîñëåäîâàòåëüíîñòåé. Äëÿ ERV I êëàññà ïðåäïîëàãàåòñÿ òåñíàÿ ñâÿçü c ðåòðîâèðóñàìè
Gammaretrovirus è Epsilonretrovirus, äëÿ ERV II êëàññà — ñ Alpharetrovirus, Betaretrovirus, Deltaretrovirus è Lentivirus, äëÿ ERV III êëàññà — ñ
Spumavirus (9).
Íà 1000 ï.í. â ðàñ÷åòíûõ ó÷àñòêàõ ÄÍÊ, ôëàíêèðîâàííûõ èíâåðòèðîâàííûì ïîâòîðîì (GA)9C è ñîîòâåòñòâóþùèõ ïî äëèíå ïîëèìîðôíûì
ôðàãìåíòàì ÄÍÊ ïðè èñïîëüçîâàíèè ýòîãî ìèêðîñàòåëëèòà â êà÷åñòâå
ïðàéìåðà â ÏÖÐ, ïðèõîäèëîñü 1,5 ó÷àñòêà ðàçëè÷íûõ ÌÃÝ. Òîëüêî â äâóõ
ôðàãìåíòàõ èç 99 èññëåäîâàííûõ îòñóòñòâîâàëè îáëàñòè ãîìîëîãèè ê ÌÃÝ.
Èíûìè ñëîâàìè, ïëîòíîñòü ëîêàëèçàöèè ó÷àñòêîâ ãîìîëîãèè ê ÌÃÝ âî
ôðàãìåíòàõ, ôëàíêèðîâàííûõ èíâåðòèðîâàííûì ïîâòîðîì (GA)9C, ñóùåñòâåííî âûøå ïðîãíîçèðóåìîé íà îñíîâàíèè ðàñïðîñòðàíåííîñòè òàêèõ
ó÷àñòêîâ â ãåíîìå ëîøàäè (5). Ïîëó÷åííûå äàííûå ñâèäåòåëüñòâóþò î òîì,
÷òî áîëüøèíñòâî ISSR-PCR ìàðêåðîâ ïðåäñòàâëåíû ôðàãìåíòàìè ÄÍÊ,
ñôîðìèðîâàííûìè ïðè ðåêîìáèíàöèÿõ ìåæäó ðàçëè÷íûìè ÌÃÝ. Ýòî
ñîîòâåòñòâóåò ðåçóëüòàòàì ñåêâåíèðîâàíèÿ ôðàãìåíòîâ ÄÍÊ ëîøàäè è
êðóïíîãî ðîãàòîãî ñêîòà, ôëàíêèðîâàííûõ èíâåðòèðîâàííûì ïîâòîðîì
ïîñëåäîâàòåëüíîñòè (GÀ)9Ñ, êîòîðûå ìû ïîëó÷èëè ðàíåå (10, 11).
Âîçìîæíûå ï ð è ÷è í û íå ï î ë íî ãî ñî â ï à ä å íè ÿ ýêñïåð è ì å í ò à ë ü í û õ è ð à ñ ÷ å ò í û õ ñ ï å ê ò ð î â ä ë ÿ ô ð à ã ì å í ò î â ÄÍÊ
Ô È Ï (GA)9C. Ñëåäóåò îòìåòèòü, ÷òî â ñèëó ðàçëè÷íûõ ïðè÷èí ïîëó÷åííûå ýêñïåðèìåíòàëüíûå ñïåêòðû ôðàãìåíòîâ ãåíîìíîé ÄÍÊ íå âñåãäà
ïîëíîñòüþ ñîîòâåòñòâóþò òåîðåòè÷åñêè îæèäàåìûì. Íàïðèìåð, ñóùåñòâóåò
âåðîÿòíîñòü òîãî, ÷òî ñåêâåíèðîâàííûå îáëàñòè íå âêëþ÷àëè àíàëèçèðóåìûé ó÷àñòîê ëèáî ñàì ñèêâåíñ îêàçàëñÿ íåïîëíûì (èçâåñòíî, ÷òî òåëîìåðíûå è öåíòðîìåðíûå ïîâòîðû èíîãäà ñåêâåíèðóþòñÿ ÷àñòè÷íî) è/èëè
ñîäåðæàë îøèáêè. Ëîêóñ ìîæåò áûòü ñâÿçàí ñ Y-õðîìîñîìîé ëîøàäè, ñåêâåíèðîâàííàÿ ïîñëåäîâàòåëüíîñòü êîòîðîé íå ïðåäñòàâëåíà â áàçå äàííûõ
(http://www.ncbi.nlm.nih.gov). Äîïîëíèòåëüíûìè ôàêòîðàìè, âåðîÿòíî, ñëóæàò ñòðóêòóðíûå îñîáåííîñòè ðàéîíà ãåíîìíîé ÄÍÊ, â êîòîðîé ëîêàëèçîâàí ôðàãìåíò ñ èíâåðòèðîâàííûì ïîâòîðîì ìèêðîñàòåëëèòà íà ôëàíãàõ.
Íàøè ðàñ÷åòíûå äàííûå ïîèñêà ïîñëåäîâàòåëüíîñòåé ÄÍÊ òîæå íå
âñåãäà ñîâïàäàëè ñ ðåçóëüòàòàìè ÏÖÐ-àìïëèôèêàöèè.  ÷àñòíîñòè, âîçíèêàëè çàòðóäíåíèÿ ïðè âûÿâëåíèè ôðàãìåíòîâ ñ äëèíîé ìåíåå 350 ï.í. íà
ýëåêòðîôîðåãðàììàõ, òîãäà êàê â ðàñ÷åòíûõ ñïåêòðàõ èõ íàõîäèëè.
Êðîìå òîãî, ÷òî îäíà ÷àñòü äàííûõ ïî ïîëèëîêóñíîìó ãåíîòèïèðîâàíèþ äîìàøíåé ëîøàäè áûëà ïîëó÷åíà íàìè ïðè èñïîëüçîâàíèè ëèîôèëèçèðîâàííûõ ÏÖÐ-íàáîðîâ PCR Core («Áèîêîì», Ðîññèÿ), äðóãàÿ — ñ ïîìîùüþ íàáîðà ÏÖÐ-Р(«Ñèíòîë», Ðîññèÿ), ñîñòîÿùåãî èç æèäêèõ ðåàãåíòîâ. Ãåíîòèïèðóÿ îäíèõ è òåõ æå æèâîòíûõ ñ ïðèìåíåíèåì â ÏÖÐ îäíîãî è òîãî æå ïðàéìåðà, ìû ðåãèñòðèðîâàëè íåîäèíàêîâûå ñïåêòðû, õà53
ðàêòåðíûå äëÿ êàæäîãî èç íàáîðîâ (ñì. ðèñ.). Ïðè÷èíà ðàçëè÷èé çàêëþ÷àëàñü â ëèîôèëèçèðîâàííîé Taq-ïîëèìåðàçå, ïðè ïðèìåíåíèè êîòîðîé
ïðåäïî÷òèòåëüíî âîñïðîèçâîäèëàñü áîëåå «ëеãêàÿ» ÷àñòü ñïåêòðà è ïðîèñõîäèëî íå òîëüêî îáùåå ñíèæåíèå ÷èñëà ëîêóñîâ, íî è ïðåâðàùåíèå íåêîòîðûõ ìèíîðíûõ ôðàãìåíòîâ ÄÍÊ â ìàæîðíûå. Ïðè äîáàâëåíèè â ðåàêöèþ æèäêîé ïîëèìåðàçû ôèðìû «Ñèíòîë» èëè «ÑèáÝíçèì» (äàííûå íå
ïðåäñòàâëåíû) ñïåêòð ïîëíîñòüþ âîññòàíàâëèâàëñÿ. Î÷åíü âàæíûì ïðåäñòàâëÿåòñÿ òî îáñòîÿòåëüñòâî, ÷òî òî÷íîñòü îòæèãà ïðàéìåðà, ïî-âèäèìîìó, íå èãðàåò â ýòîì ïðîöåññå îïðåäåëÿþùåé ðîëè. Ïîäòâåðæäåíèåì
òîìó ñëóæèò ïîëíîå îòñóòñòâèå â ñïåêòðàõ ñ ëèîôèëèçèðîâàííîé Taq-ïîëèìåðàçîé ôðàãìåíòà 877 ï.í. ñ ñàìûìè òî÷íûìè ñàéòàìè îòæèãà ñðåäè
âñåõ íàéäåííûõ ïîñëåäîâàòåëüíîñòåé, ïðè ýòîì íåêîòîðûå ëîêóñû, èìåþùèå äàæå áîëüøóþ äëèíó, âîñïðîèçâîäèëèñü ñòàáèëüíî (ñì. ðèñ., Á).
 ñïåêòðå àìïëèêîíîâ, ïîëó÷åííûõ ñ ëèîôèëèçèðîâàííîé Taqïîëèìåðàçîé, îáíàðóæèâàëñÿ ìèíîðíûé êîìïîíåíò äëèíîé ïðèìåðíî
310 ï.í., äëÿ êîòîðîãî òàêæå áûëî íàéäåíî ñîîòâåòñòâèå â ñèêâåíñàõ õðîìîñîì (ñì. òàáë. 2). Ïðè èñïîëüçîâàíèè íàáîðà ÏÖÐ-ÐÂ âûÿâèòü ýòîò
ôðàãìåíò íå óäàëîñü. Â òî æå âðåìÿ ñ ïîìîùüþ íàáîðà ÏÖÐ-ÐÂ ïðè ïîâûøåíèè òåìïåðàòóðû îòæèãà èç êëàñòåðà 800-900 ï.í. âûäåëÿëñÿ ëîêóñ
ðàçìåðîì 880 ï.í., ÷òî ñîîòâåòñòâóåò ðàñ÷åòíîìó ôðàãìåíòó ÄÍÊ äëèíîé
877 ï.í., èìåþùåìó íà ôëàíãàõ ïîñëåäîâàòåëüíîñòè ñ âûñîêîé êîìïëåìåíòàðíîñòüþ ê ïðàéìåðó (GA)9C.
Íåêàíîíè÷åñêèå ñòðóêòóðû ÄÍÊ â ðàñ÷åòíûõ è ýêñïåð è ì å í ò à ë ü í û õ ñ ï å ê ò ð à õ ô ð à ã ì å í ò î â Ä Í Ê Ô È Ï (GA)9 C. Êàê îò6. Õàðàêòåðèñòèêà ïîòåíöèàëüíûõ øïèëå÷íûõ ìå÷àëîñü, ïðè÷èíîé íåïîëñòðóêòóð âî ôëàíêèðîâàííûõ èíâåðòèðîâàí- íîãî ñîâïàäåíèÿ ðàñ÷åòíûõ
íûì ïîâòîðîì (GA)9C ïîñëåäîâàòåëüíîñòÿõ è ýêñïåðèìåíòàëüíûõ ñïåêñåêâåíèðîâàííîãî ãåíîìà ó äîìàøíåé ëîøàäè òðîâ ISSR-PCR ìàðêåðîâ ìîEquus caballus
ãóò áûòü îñîáåííîñòè âíóòÄëèíà Äëèíà
×èñëî øïè- Äîëÿ G-C ïàð ðåííåé ñòðóêòóðû àíàëèçèðóåìûõ ôðàãìåíòîâ ÄÍÊ, â
ëîêóñà, ôðàãìåí- Õðîìîñîìà ëåê âíóòðè â âîçìîæíûõ
ï.í.
òà, ï.í.
øïèëüêàõ, %
ëîêóñà
÷àñòíîñòè ïðåäðàñïîëîæåí900
900
24-ÿ
7
25,0-2,5
íîñòü ê ôîðìèðîâàíèþ íå800-900 876
Õ
9
0-50,0
800-900 871
18-ÿ
4
25,0-100
êàíîíè÷åñêèõ âòîðè÷íûõ
800-900 857
3-ÿ
1
62,5
ñòðóêòóð. Ñàìî èõ ïðèñóò800-900 832
6-ÿ
4
50,0-62,5
ñòâèå íà ôëàíãàõ èíâåðòè800-900 823
19-ÿ
2
37,5
550
637 (552)
Õ
5
22,2-50,0
ðîâàííîãî ïîâòîðà ìèêðî550
572 (550)
10-ÿ
1
37,5
ñàòåëëèòà ñâèäåòåëüñòâóåò
500
494
1-ÿ
1
37,5
490
489
28-ÿ
4
37,5-75,0
î âåðîÿòíîñòè îáðàçîâàíèÿ
380
378
11-ÿ
1
50,0
øïèëåê, ÷òî ìîæåò ñïîñîá380
377
21-ÿ
0
–
360 (370) 364
5-ÿ
0
–
ñòâîâàòü ïîâûøåíèþ ãåíîì310
309
5-ÿ
0
–
íîé íåñòàáèëüíîñòè â òàêèõ
Ï ð è ì å ÷ à í è å. Ïðåäñòàâëåíû äàííûå äëÿ ëîêóñîâ äëèíîé äî
2000 ï.í., ñîîòâåòñòâóþùèõ ïî ðàçìåðó ôðàãìåíòàì ÄÍÊ, àì- ó÷àñòêàõ (4).  ýòîé ñâÿçè
ïëèôèöèðîâàííûì â ISSR-PCR ñ ïîñëåäîâàòåëüíîñòüþ (GA)9C â ìû ïðîàíàëèçèðîâàëè âîçêà÷åñòâå ïðàéìåðà. Ïðî÷åðêè îçíà÷àþò îòñóòñòâèå òàêèõ ïîñëåäîâàòåëüíîñòåé. Ìèíèìàëüíîå ÷èñëî ñïàðåííûõ îñíîâàíèé äëÿ ìîæíîñòè äëÿ ôîðìèðîâàôîðìèðîâàíèÿ øïèëåê — 8.
íèÿ øïèëåê (äëèíîé íå ìåíåå 8 ï.í.) è G4 êâàäðóïëåêñîâ (ñòðóêòóðíûå ýëåìåíòû ñ ïîâûøåííîé òåðìîóñòîé÷èâîñòüþ, îáðàçóþùèå ÷åòûðåõöåïî÷å÷íûå ñòðóêòóðû, â êîòîðûõ ãóàíèíû ôîðìèðóþò
ïëîñêîñòü áëàãîäàðÿ ïîïåðå÷íûì ñâÿçÿì) âíóòðè àìïëèêîíîâ (12). Ðàññìîòðåíû ñâÿçè ìåæäó ïëîòíîñòüþ ëîêàëèçàöèè òàêèõ ñòðóêòóð â ðàñ÷åòíûõ
ñïåêòðàõ è ïîëèìîðôèçìîì ôðàãìåíòîâ ÄÍÊ ñîîòâåòñòâóþùåé äëèíû â
ýêñïåðèìåíòàëüíûõ ñïåêòðàõ ó ëîøàäåé (ñì. òàáë. 5, 6).
54
Îêàçàëîñü, ÷òî â ïîñëåäîâàòåëüíîñòè äëèíîé 877 ï.í., ëîêàëèçîâàííîé â Õ-õðîìîñîìå, âûÿâëÿëîñü 9 äîñòàòî÷íî êðóïíûõ øïèëåê, ÷òî
áîëüøå, ÷åì âî âñåõ îñòàëüíûõ ó÷àñòêàõ (äëÿ àíàëèçà èñïîëüçîâàëè îëèãîêàëüêóëÿòîð (http://www.bio.bsu.by/molbiol/oligocalc.html).  ïîñëåäîâàòåëüíîñòè äëèíîé 871 ï.í. èç 18-é õðîìîñîìû îáíàðóæèëèñü 4 ïîòåíöèàëüíûå øïèëüêè. Âàæíî ïîä÷åðêíóòü, ÷òî â ýêñïåðèìåíòàëüíûõ ñïåêòðàõ
ó÷àñòêè áëèçêîé äëèíû âûÿâëÿëèñü òîëüêî ïðè èñïîëüçîâàíèè íàáîðà
ÏÖÐ-ÐÂ. Â òî æå âðåìÿ äëÿ íàèáîëåå âîñïðîèçâîäèìûõ â ðàçíûõ óñëîâèÿõ
ÏÖÐ ôðàãìåíòîâ ÄÍÊ áûëà õàðàêòåðíà ïîíèæåííàÿ ÷àñòîòà òàêèõ ïîñëåäîâàòåëüíîñòåé. Íàïðèìåð, ôðàãìåíò äëèíîé 380 ï.í. èìåë âñåãî îäíó
øïèëüêó, ïðè÷åì â íàèáîëåå áëèçêîì ïî äëèíå ôðàãìåíòå äëèíîé 377 ï.í.
(21-ÿ õðîìîñîìà) ïîòåíöèàëüíûå øïèëüêè îòñóòñòâîâàëè. Íå èìåëà ïîòåíöèàëüíûõ øïèëåê è íóêëåîòèäíàÿ ïîñëåäîâàòåëüíîñòü ìèíîðíîãî ëîêóñà äëèíîé 310 ï.í. (309 ï.í., 5-ÿ õðîìîñîìà), àìïëèôèöóðóåìîãî â ýêñïåðèìåíòå ïðè èñïîëüçîâàíèè ëèîôèëèçèðîâàííîé Taq-ïîëèìåðàçû. Ïîëó÷åííûå äàííûå ïîçâîëÿþò ïðåäïîëàãàòü, ÷òî ïðåäðàñïîëîæåííîñòü ê
ôîðìèðîâàíèþ âòîðè÷íûõ ñòðóêòóð ìîæåò âëèÿòü íà âîñïðîèçâîäèìîñòü
ôðàãìåíòîâ ÄÍÊ â ðàçíûõ óñëîâèÿõ ÏÖÐ.
Èçâåñòíî, ÷òî G4 êâàäðóïëåêñû òåñíî ñâÿçàíû ñ âîçíèêíîâåíèåì
äâóöåïî÷å÷íûõ ðàçðûâîâ ÄÍÊ (DSBs) (13). Èìåþòñÿ äàííûå, ñâèäåòåëüñòâóþùèå î âûðàæåííûõ àññîöèàöèÿõ ìåæäó íàëè÷èåì G4 êâàäðóïëåêñîâ è
äâóöåïî÷å÷íûìè ðàçðûâàìè ÄÍÊ (DSBs) â ìèòîòè÷åñêèõ è ìåéîòè÷åñêèõ
êëåòêàõ (14).  ýòîé ñâÿçè ìû ñîïîñòàâèëè ÷àñòîòó ïîòåíöèàëüíûõ G4
êâàäðóïëåêñîâ â ðàñ÷åòíûõ ñïåêòðàõ ñåêâåíèðîâàííûõ íóêëåîòèäíûõ ïîñëåäîâàòåëüíîñòåé, áëèçêèõ ïî äëèíå ê êîíñåðâàòèâíûì è ïîëèìîðôíûì
ôðàãìåíòàì ÄÍÊ èç ýêñïåðèìåíòàëüíûõ ñïåêòðîâ ISSR-PCR ìàðêåðîâ
(http://bioinformatics.ramapo.edu/QGRS/index.php). Ïðè ýòîì âûðàæåííûõ
ðàçëè÷èé ìû íå îáíàðóæèëè (ñì. òàáë. 5, 6).  îáåèõ ãðóïïàõ G4 êâàäðóïëåêñû âñòðå÷àëèñü ïðèìåðíî ñ ðàâíîé ÷àñòîòîé (îêîëî 3,4 ïîòåíöèàëüíîãî G4 êâàäðóïëåêñà íà 1000 ï.í.). Òåì íå ìåíåå, îäèí èç ðàñ÷åòíûõ ó÷àñòêîâ, ïî äëèíå íàèáîëåå áëèçêèé ê âûñîêîïîëèìîðôíîìó â ýêñïåðèìåíòàëüíûõ ñïåêòðàõ ôðàãìåíòó ÄÍÊ äëèíîé 550 ï.í., ïðîÿâëÿë âûðàæåííóþ ïðåäðàñïîëîæåííîñòü ê ôîðìèðîâàíèþ òàêèõ ñòðóêòóð (ñì. òàáë.
5). Áîëåå òîãî, â ãåíîìå ÷åëîâåêà, íàïðèìåð, àíàëîãè÷íûå ïîñëåäîâàòåëüíîñòè â ñðåäíåì âñòðå÷àþòñÿ ñ ÷àñòîòîé ïðèìåðíî 0,125 ïîòåíöèàëüíîãî
G4 êâàäðóïëåêñà íà 1000 ï.í. (15).  ïîëó÷åííûõ íàìè ðàñ÷åòíûõ ïîñëåäîâàòåëüíîñòÿõ àìïëèêîíîâ ISSR-PCR ñïåêòðà ïî ïðàéìåðó (GA)9Ñ ýòà
öèôðà ïðèìåðíî â 27 ðàçà áîëüøå, ÷òî ìîæåò ñâèäåòåëüñòâîâàòü î ïîâûøåííîé ÷àñòîòå ëîêàëèçàöèè G4 êâàäðóïëåêñîâ â ó÷àñòêàõ ãåíîìà, ðàñïîëîæåííûõ ìåæäó èíâåðòèðîâàííûìè ïîâòîðàìè ìèêðîñàòåëëèòîâ.
Èòàê, ó äîìàøíåé ëîøàäè èíâåðòèðîâàííûå ïîâòîðû ìèêðîñàòåëëèòíûõ ëîêóñîâ, ôëàíêèðóþùèå ó÷àñòêè ÄÍÊ äëèíîé äî 2000 ï.í. (ISSRPCR ìàðêåðû), ïðåèìóùåñòâåííî ñôîðìèðîâàëèñü â ðåçóëüòàòå ñëîæíûõ
ðåêîìáèíàöèîííûõ ñîáûòèé ìåæäó ìîáèëüíûìè ãåíåòè÷åñêèìè ýëåìåíòàìè, ïðèíàäëåæàùèìè ê ðàçíûì êëàññàì òðàíñïîçîíîâ. Ïîëèìîðôíûå
àìïëèêîíû ÄÍÊ, ôëàíêèðîâàííûå èíâåðòèðîâàííûì ïîâòîðîì (GA)9Ñ,
êîòîðûå ïî äëèíå áëèçêè ê îáíàðóæåííûì â ñåêâåíèðîâàííûõ ãåíîìíûõ
ïîñëåäîâàòåëüíîñòÿõ, ìîãóò ñîäåðæàòü áîëüøå ó÷àñòêîâ ãîìîëîãèè ñ ðåòðîòðàíñïîçîíàìè (â ÷àñòíîñòè, ñ ïîñëåäîâàòåëüíîñòÿìè ýíäîãåííîãî ðåòðîâèðóñà III êëàññà — ERV III), ÷åì êîíñåðâàòèâíûå ôðàãìåíòû. Íàëè÷èå
â ISSR-PCR ìàðêåðå ïðîäóêòîâ ðåêîìáèíàöèé ñ ðåòðîòðàíñïîçîíàìè (â
îòëè÷èå îò ÄÍÊ-òðàíñïîçîíîâ), ïî âñåé âèäèìîñòè, àññîöèèðîâàíî ñ ïî55
âûøåííûì ïîëèìîðôèçìîì ýòîãî ãåíîìíîãî ó÷àñòêà. Îáðàçîâàíèå âòîðè÷íûõ ñòðóêòóð âíóòðè ïîñëåäîâàòåëüíîñòè ÄÍÊ, ôëàíêèðîâàííîé èíâåðòèðîâàííûì ïîâòîðîì ìèêðîñàòåëëèòà, ñïîñîáíî ïîâëèÿòü íà âîñïðîèçâîäèìîñòü ïîëèëîêóñíîãî ãåíîòèïèðîâàíèÿ ïî ISSR-PCR ìàðêåðàì. Â
ó÷àñòêàõ ãåíîìíîé ÄÍÊ äëèíîé äî 2000 ï.í., ôëàíêèðîâàííûõ èíâåðòèðîâàííûì ïîâòîðîì (GA)9C, îáíàðóæèâàåòñÿ ïîâûøåííàÿ ïëîòíîñòü íóêëåîòèäíûõ ïîñëåäîâàòåëüíîñòåé, ïîòåíöèàëüíî ïðåäðàñïîëîæåííûõ ê ôîðìèðîâàíèþ G4 êâàäðóïëåêñîâ, ÷òî, âåðîÿòíî, ìîæåò áûòü îáóñëîâëåíî ïîâûøåííîé àêòèâíîñòüþ ðåêîìáèíàöèé â ýòèõ ó÷àñòêàõ.
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1ÔÃÁÎÓ
ÂÏÎ Ðîññèéñêèé ãîñóäàðñòâåííûé
àãðàðíûé óíèâåðñèòåò—ÌÑÕÀ èì. Ê.À. Òèìèðÿçåâà,
127550 Ðîññèÿ, ã. Ìîñêâà, óë. Òèìèðÿçåâñêàÿ, 49,
e-mail: [email protected];
56
Ïîñòóïèëà â ðåäàêöèþ
31 ìàðòà 2014 ãîäà
2ÃÍÓ
Öåíòð ýêñïåðèìåíòàëüíîé ýìáðèîëîãèè
è ðåïðîäóêòèâíûõ áèîòåõíîëîãèé,
127422 Ðîññèÿ, ã. Ìîñêâà, óë. Êîñòÿêîâà, 12, ñòð. 4,
e-mail: [email protected]
ISSR-PCR MARKERS AND MOBILE GENETIC ELEMENTS
IN HORSE (Equus caballus) GENOME
N.V. Bardukov1, A.V. Feofilov1, T.T. Glazko1, 2, V.I. Glazko1, 2
1K.A.
Timiryazev Russian State Agrarian University, 49, ul. Timiryazeva, Moscow, 127550 Russia, e-mail vglazko@
yahoo.com;
2Center for Experimental Embryology and Reproductive Biotechnology, str. 4, 12, ul. Kostyakova, Moscow, 127422
Russia, e-mail [email protected]
Received March 31, 2014
doi: 10.15389/agrobiology.2014.4.42eng
Abstract
The patterns and causes of the differences in the polymorphism of individual microsatellites, and species specificity of this variability are still not enough studied. Test systems developed by
the International Association of Animal Genetics (ISAG) for genetic certification of agricultural
animals, assessment of interbreed relations, the level of animal group consolidation, include a small
number of genetic markers. The use of microsatellite loci fragments as PCR primers for polylocus
genotyping and genome scan of agricultural animal species is more simple and effective methodical
approach. We proposed a method for identifying positioning of microsatellite inverted repeat areas in
genome sequences and for comparisons of this calculated areas to experimental DNA spectra amplified in PCR with the use of microsatellite loci as primers (ISSR-PCR markers). Here we report the results of the comparisons carried out for domestic horses (Equus caballus) of Altai breed
(n = 96), Trotter breed (n = 48), Karachai breed (n = 34). The DNA fragments (99 sites) in the
sequenced genome of the domestic horse, flanked by inverted repeat (GA)9C, were revealed and the
presence of homologous sequences to mobile genetic elements was analyzed. The nucleotide sequences were examined in the DNA fragments (expected size/obtained size) of different location:
1250-1350/1326 bp (chromosome 17), 1250-1350/1302 bp (chromosome 2), 1250-350/1296 bp (Õchromosome), 1250-1350/1287 bp (chromosome 20), 980/986 bp (chromosome 23), 800-900/900 bp
(chromosome 24), 800-900/876 bp (Õ-chromosome), 800-900/871 bp (chromosome 18), 800900/857 bp (chromosome 3), 800-900/832 bp (chromosome 6), 800-900/823 bp (chromosome 19),
740/739 bp (chromosome 16), 580/585 bp (chromosome 1), 550/637 bp (Õ-chromosome), 550/572
bp (chromosome 10), 500/494 bp (chromosome 1), 490/489 bp (chromosome 28), 380/378 bp
(chromosome 11), 380/377 bp (chromosome 21), 360 (370)/364 bp (chromosome 5), 310/309 bp
(chromosome 5). The PIC (polymorphic information content) values for loci were calculated. In the
representatives of several breeds, the conservative and polymorphic DNA fragments were observed in
the amplification product spectra obtained in the polymerase chain reaction with a primer sequence
(GA)9C. It was turned out that the polymorphous DNA fragments in different animals, in most
cases, contained the homologous sequences to fragments of retrotransposons (to ERV III, particularly), while the conservative ones contained homologous sequences to DNA transposons. An increased density of nucleotide sequences, potentially prone to the G4 quadruplex formation, was observed in the studied horse genomic DNA fragments, flanked by the inverted repeat (GA)9C, that
could be associated with an increased recombination in these areas. A secondary structure in the
DNA regions flanked by a microsatellite repeat can influence the results of genotyping in case the
ISSR-PCR markers are used. The data obtained suggest the relationship between the polymorphism
of DNA fragments, flanked by inverted repeats of microsatellites, and a mobility of retrotransposons.
Keywords: ISSR-PCR, anonymous DNA markers, Equus caballus, DNA transposons,
retrotransposons, noncanonical DNA structures.
Íîâûå êíèãè
Ñ ò ó ä å í ö î â À.Ï., Ø è ï è ë î â Â.Ñ.,
Í è ê è ò è í Â.ß. è äð. Àêóøåðñòâî, ãèíåêîëîãèÿ è áèîòåõíèêà ðåïðîäóêöèè æèâîòíûõ.
Ì.: èçä-âî «ÊîëîñÑ», 2012, 439 ñ.
Ðàññìîòðåíû àíàòîìèÿ è ôèçèîëîãèÿ îðãàíîâ ðàçìíîæåíèÿ, ñïîñîáû îñåìåíåíèÿ æèâîòíûõ, ôèçèîëîãèÿ è ïàòîëîãèÿ áåðåìåííîñòè, ðîäîâ è ïîñëåðîäîâîãî ïåðèîäà.
Èçëîæåíû âîïðîñû èììóíîëîãè÷åñêèõ âçàèìîîòíîøåíèé ïðè îïëîäîòâîðåíèè è áåðå-
ìåííîñòè, âçàèìîñâÿçè íåðâíîé, ýíäîêðèííîé è èììóííîé ñèñòåì â ðåãóëÿöèè ãîìåîñòàçà. Äàíû ñâåäåíèÿ ïî àêóøåðñêîé òåðàïèè, áîëåçíÿì ìîëî÷íîé æåëåçû, ãèíåêîëîãèè äîìàøíèõ æèâîòíûõ. Âíåñåíû èçìåíåíèÿ è äîïîëíåíèÿ â ðàçäåëû òðàíñïëàíòàöèè
çàðîäûøåé, ôèçèîëîãèè ðàçìíîæåíèÿ ïóøíûõ çâåðåé, èìïîòåíöèè ïðîèçâîäèòåëåé,
ïðîôèëàêòèêè áåñïëîäèÿ æèâîòíûõ è ò.ä.
Äëÿ ñòóäåíòîâ âóçîâ, îáó÷àþùèõñÿ ïî ñïåöèàëüíîñòÿì «Âåòåðèíàðèÿ» è «Çîîòåõíèÿ».
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